CN1299411A - Hyperthermic inducible expression vectors for gene therapy and methods of use thereof - Google Patents

Abstract

Methods and compositions are provided for transgene expression in target cells. Expression constructs using an inducible amplification system to drive expression of a therapeutic gene or other gene of interest in mammalian host cells are provided, as well as methods therefor. Inducible expression of the transgenes at high levels under physiologic conditions results from induction by hyperthermic conditions relative to the basal temperature of the host cells.

Description

The high temperature induction expression vector and the using method thereof that are used for gene therapy

The technical field of the invention

The present invention relates to field of gene, relate in particular to the method and composition that improves transgene expression.

Prior art related to the present invention

People expect that gene therapy can be widely used in treating a series of cancers and other a large amount of diseases.Virus vector is a kind of transgene carrier that uses at present.People have made up many virus expression systems and they have been changed foreign gene over to somatic ability and assessed.Especially, for the carrier system based on retrovirus and adenovirus, people have carried out the further investigation more than 10 years.Recently, adeno-associated virus (AAV) has been showed the present retrovirus more commonly used of replacement and the possibility of adenovirus carrier to people.The fat carrier that comprises cationic lipid and liposome also can be used for transforming the plasmid DNA that contains therapeutic gene.

The process of gene therapy disease and pathology comprises that gene changes over to and the new stable or temporary transient insertion cell of genetic information.Show by the test that the normal allele with the gene of the function coding wanting to obtain is changed again over to the hereditary defect of coming suppressor gene in the body, this method of gene therapy is feasible (Rosenberg etc., New Eng.J.Med_323:570 (1990)) clinically.In fact, clinically lay a good foundation for the relevant difficult problem of the gene therapies such as potentially dangerous that solve genetic transformation efficiency, gene expression regulation, use virus vector the early stage that the coverage of carrying out is very wide at present with clinical study about genetic diseases.Most of is that the gene therapy test of carrier is to test at the external transformed target cell of doing earlier with virus, and then carries out in vivo test.Virus vector also can directly carry out in vivo test, but use can induce neutral antibody repeatedly.

The subject matter that potential clinical gene therapy faces is how to make a large amount of clinical expression of foreign gene in the specific cells tissue of human body.Gene is adjusted element for the possible method that provides is provided.Gene adjustment element such as promotor and enhanser suppress cell specificity activity, and they can be activated by respective element by some specificity factors.Use adjustment element such as promotor and come promotor gene to express, the controlled and selective expression of foreign gene in the vector construction body become easily, for example can make the heat-inducible promoter expression alien gene by heat shock.

United States Patent (USP) 5,614,381; 5,646,010, and PCT publication application WO89/00603 is higher than 42 ℃ heat shock about use to induce transgene expression.This temperature is infeasible in the human gene therapy, and it is impossible human body not being damaged because continue this temperature for some time.

Gene therapy can comprise that traditional cancer treatment methods such as cytotoxic drug and radiation treatment unite use with some other.There are some researches show (Harisiadis etc., Cancer, 41:2131-2142 (1978)), high temperature can strengthen the cell killing effect of ray, particularly tumour cell in animal body, and this has improved the result in the randomized clinical trial.Yet, the subject matter that hyperthermia treatment faces be it effectively heat shock is large-scale and go deep into intravital tumour.

Therefore, exploitation can be at 42 ℃ or the carrier that more uses under the low temperature, by in the selectable inductive treatment expression of gene of body part, patient is carried out system or localized treatment, will be of great use.

The present invention's general introduction

The invention provides method how to control the abduction delivering of polypeptide in cell.It also provides the use of the heat-inducible promoter in the method for controlling the abduction delivering of poly-nucleotide in the mammalian cell especially.The present invention can overcome the defective of mentioning in the correlation technique of front at 42 ℃ or the heat shock control carrier that uses of low temperature more by providing.These methods are treated patient by the abduction delivering of therapeutic gene.

In according to one embodiment of present invention, provide a kind of expression method of transgenosis in mammalian cell of controlling.This method at first provides an expression vector, and this carrier comprises two portions: (ⅰ) inducible promoters that links to each other with a coding Reverse Activity factor, second promotor that is connected with selected poly-nucleotide (therapeutic gene) with (ⅱ).The Reverse Activity factor activator that second promotor is expressed by same construct.This method has also comprised the step that expression construct is changed over to cell.At last, make cell be in the state that evoked promoter is activated and begin to express selected protein.

According to a preferred embodiment of the present invention, first inductive promotor is a heat-inducible promoter, and its activation condition is a high temperature.This pyritous scope is to about 42 ℃ from base temperature.In use, base temperature is defined as cell temperature in the raw, and for example, the base temperature of mammiferous epidermic cell is 33 ℃, yet lung may be 39 ℃.Under special circumstances, the use of high temperature induction carrier comprises the temperature of cell is raise from base temperature that the most frequently used is to be elevated to about 42 ℃ from 37 ℃.The condition of high temperature can be from 37 ℃ to about 41 ℃, perhaps from 39 ℃ to about 40 ℃.Heat-inducible promoter is from one group of heat shock protein promotor HSP70, HSP90, and HSP27, HSP27, HSP73, HSP25 derives out among the HSP28.This generally starting also can be used as evoked promoter in expression construct.A minimal promoter that develops from heat-inducible promoter HSP70 and comprise that the HSP70B promotor of about 400bp can be selected to the present invention.

In according to another embodiment of the invention, evoked promoter comprises a hypoxemia reactive element (HRE).This reactive element comprises the binding site of a hypoxia inducible factor-I (HIF-I) at least.

In according to one embodiment of present invention, second promotor can comprise human immunodeficiency virus-1 (HIV-1) promotor and human immunodeficiency virus-2 (HIV-2) promotor from one group screens.In according to other embodiments of the invention, trans-activating factor is an activating transcription factor (TAT).

Specific poly-nucleotide can encode an albumen or one section polypeptide.For example, the poly-nucleotide of selecting following any one albumen of to encode, the ornithine decarboxylase antizyme, p53, p16, neural differentiation factor, interleukin 1 (IL1), interleukin II (IL2), interleukin 4 (IL4), interleukin 7 (IL7), interleukin 12 (IL12), Interleukin-15 (IL15), FLT-3 part, granular leukocyte macrophage stimulus factor (CM-CSF), granulocyte colony-stimulating factor (G-CSF), gamma-interferon (IFN γ), alpha-interferon (IFN α), tumour necrosis factor (TNF), simple luxuriant exanthema virus thymidine kinase (HSV-TK), I-CAM1, human leucocyte antigen (HLA)-B7 (HLA-B7), or metalloprotease tissue suppresses son (TIMP-3).In this case, specific nucleic acid is positioned at the responsive initiator by second promotor control.

Under the another kind of situation, the expression of selected nucleic acid is only transcribed and not translation, and it produces ribozyme.In this case, selected nucleic acid also is the responsive initiator that is positioned at by second promotor control.

Under another situation, the expression of selected nucleic acid is only transcribed and not translation, and it produces the RNA molecule of adjusting the performance function as antisense.In this case, selected nucleic acid can be target gene or function fragment, and it is arranged in the expression construct in the antisense initiator district relevant with described second promotor.

Expression construct can also comprise a gene that the screening sign is encoded, the plain resistance of the certain kind of berries that has a tidal wave of, the plain resistance of the new certain kind of berries, the plain resistance of the purine certain kind of berries, red certain kind of berries element, gpt, DHFR, green fluorescent protein or histidinol.Expression construct also can comprise the second specific poly-nucleotide that (ⅰ) is connected with second promotor, and (ⅱ) the internal ribosome binding site between first and second promotors.

Target cell can be a tumour cell, is positioned at the cell of inside tumor, perhaps the intravital cell of Mammals.Expression construct is gone into cell can be in vivo or external finishing.In one embodiment, the importing of construct is by the control of transhipment instrument, and this transhipment instrument is from liposome, retrovirus, and adenovirus, adeno-associated virus, beans shape virus, simple luxuriant exanthema virus, or screen in the poxvirus.

In according to another embodiment of the invention, provide to make transgene reach the method that treatment requires.This method at first provides one first expression construct, it comprises an evoked promoter that links to each other with trans activating transcription factor, this method also provides one second expression construct, this construct comprises second promotor that links to each other with the second specific polypeptide, and this second promotor is activated by the trans-activating factor of encoding in first expression construct.First and second expression construct change in the target cell simultaneously, make target cell be in evoked promoter activated state simultaneously, and specific like this nucleic acid just begins abduction delivering.In a preferred embodiment, first and second expression construct are positioned on the same carrier.And evoked promoter heat-inducible promoter normally, its activation condition is to be lower than 42 ℃ and the inducing temperature that is higher than normal body temperature.

One or two expression construct are changed over to cell can be in vivo or external finishing.The expression product of selected nucleic acid is to the toxic effect of the intravital pathogenic agent of target, and these pathogenic agent comprise virus, bacterium, fungi or parasite.In some cases, expression product can suppress the growth of target cell.In another embodiment of the present invention, expression product has replaced the intravital deficient protein of target.Expression product sometimes can also neuroactive regeneration.

In other embodiment according to the present invention, provide treatment to comprise the method for people's mammalian cancer, its step comprises: an expression construct (a) is provided, this construct comprises (ⅰ) evoked promoter, heat-inducible promoter normally, it links to each other with the gene of coding trans-activating factor, (ⅱ) second promotor that is connected with selected nucleic acid, and it is activated by trans-activating factor; (b) this expression construct is imported tumour cell; And (c) make tumour cell be in evoked promoter activated state, selected polynucleotide are expressed to suppress the growth of tumour cell with sufficiently high quantity.If evoked promoter is a heat-inducible promoter, its inductive condition is to be lower than 42 ℃ and the temperature that is higher than normal body temperature so.

This methods of treatment can be united use with some known treatment method for cancer, and these methods comprise: outside beamtherapy, brachytherapy, chemotherapy and operation.Treatable cancer comprises: brain, lung, liver, spleen, kidney, lymph, small intestine, pancreas, hemocyte, stomach, breast, uterus, prostate gland, testis, oral cavity, perineum, skin, head and neck, oesophagus, marrow and leukemia etc.

In one particular embodiment of the present invention, special genes is the ornithine decarboxylase antizyme.After making cell be in evoked promoter activated state, handle tumour cell with antiradiation drug WR-33278 or WR-1065, last, come the kill tumor cell with ray.

The present invention also provides the activation Mammals, for example people's immunoreactive method.The activated immune response comprises humoral immunization and cellular immunization.In one embodiment, this method comprises: an expression construct (a) is provided, and this construct comprises (ⅰ) evoked promoter, heat-inducible promoter normally, and it links to each other with the gene of a coding trans-activating factor; And second promotor that (ⅱ) is connected with specific nucleic acid, it is activated by trans-activating factor; (b) expression construct is imported mammalian cell; And (c) regulate cell state activation-inducing promotor, specific gene is efficiently expressed activate mammiferous immunity system.If this evoked promoter is a heat-inducible promoter, its activation condition is one and is higher than basal temperature and is lower than 42 ℃ temperature so.

In another embodiment, the activated immune response is to kill the mammalian cell that those contain expression construct.This method also relates to some sophisticated treatment method for cancer, and these methods comprise: chemotherapy, outside beamtherapy, brachytherapy, and operation.

In another embodiment, provide an expression construct, this construct comprises: (a) gene to the trans-activating factor coding; (b) evoked promoter that is connected with this gene; (c) selected poly-nucleotide; And second promotor that (d) is connected with this nucleic acid.Second promotor in the construct is activated by trans-activating factor.In a preferred embodiment, evoked promoter is a heat-inducible promoter, and selected poly-nucleotide can be by abduction delivering under the condition of high temperature, and this high temperature is between 37 ℃ and 42 ℃.In an optional embodiment, evoked promoter also comprises the hypoxemia reactive element.This expression construct also can comprise one second selected poly-nucleotide, and this second selected poly-nucleotide also is connected with second promotor, and by an IRES itself and the first selected poly-nucleotide is separated.

The invention provides the cell that comprises expression construct.The expression construct that is provided can be used in the method that changes mammiferous genetic material.

Other purpose of the present invention, characteristic and advantage elaborate chapters and sections below.It should be understood that the description about embodiments of the invention only is a kind of elaboration, on the basis of grasping marrow of the present invention and scope, suitable variation of the present invention and modification are still belonged to category of the present invention.Brief description of drawings

Following diagram is the part of this explanation, and it further sets forth characteristics more of the present invention.People can understand the present invention better in conjunction with diagram and the following detailed explanation to embodiment.

Fig. 1 shows about the active underlying carrier of quantitative analysis heat-inducible promoter.This plasmid comprises a minimal promoter that comes from the evolution of HSP70B promotor.A reporter gene, as strengthening fluorescin (EGFP), β-gal, or IL-2 is inserted in the multiple clone site, so that its expression is subjected to the control of basic HSP70B promotor.This plasmid also comprises plain resistance of the new certain kind of berries and the plain resistant gene of the blue or green certain kind of berries of peace benzyl, and the screening in the microbial culture system of mammal cell line and standard is necessary for this plasmid for they.The S8 plasmid comprises the plasmid that shows with the EGFP that inserts multiple clone site.

Fig. 2 has shown fluorescence activated cell letter sorting (FACS) column diagram of the stable DU-145 cell that has changed the S8 plasmid over to.Fluorescence from left to right strengthens, and top column diagram is the DU-145 cell that has changed S8 over to, but does not pass through heat shock.Following column diagram is at one hour the DU-145 cell that changes success over to of 42 ℃ of following heat shocks.

Fig. 3 shows the FACS column diagram of the MCF7 clone of three different S8 transfections.The MCF7 cell that has changed the S8 construct over to is classified according to FACS.The clone that initiating cell system is selected from a polyclone.Activated clone (promptly expressing the cell of EGFP) and non-activating cells system are separately.After separating once, cultivate positive strain and it is screened once more to obtain purer positive cell line.In this example, polyclone positive cell line MCF7-S8-PS2 is the product of twice screening on the MCF7-S8-P basis.

Fig. 4 shows is expression by EGFP in the different clones of facs analysis.Clone at first changes the S8 plasmid over to, cloned cell line or cultivation polyclone clone.In some cases, the expression by facs analysis EGFP comes sorting.To the quantitatively also mapping of total fluorescence mean value.

Fig. 5 has shown the expression of the EGFP of the stable DU-145 clone (DU-S8-PS2) that changes over to that twice heat shock screened.DU-S8-PS2 clone is 40 ℃ or 42 ℃ of heat shocks and recover several times, then by the facs analysis cell.

Fig. 6 has shown that the stable transformed cells through recovering 16 hours after the heat shock is the EGFP expression of DU-145.One of them clone (DU-S8-PS2) has changed the S8 plasmid over to, and another clone (DU-V9-PS2) has changed the V9 plasmid over to, and what the difference of V9 plasmid and S8 plasmid was the V9 plasmid is to link to each other with the CMV promotor rather than link to each other with the HSP70B (see figure 7).Clone is with different temperature heat shocks and it was recovered 16 hours.The DU-145 clone that does not change plasmid in contrast.

Fig. 7 has shown the analysis column diagram of the V9 plasmid that contains the MCV promotor that is connected with enhancing fluorescin (EGFP).

What Fig. 8 showed is the underlying carrier that comprises second promotor of amplifying the heat shock effect.This plasmid comprises a multiple clone site (MCS) that is subjected to the HSP70B regulation and control, and it also comprises a therapeutic gene by the control of second promotor.This plasmid also includes the new certain kind of berries plain resistance, ampicillin resistance gene and the leading element of growing in bacterium.In the pC8 plasmid, its second promotor is the terminal repetition fragment of length (LTR) of HIV-1, and therapeutic gene is IL2.In plasmid pf12, inserted the tat gene among the MCS, second promotor is the LTR of HIV-I, therapeutic gene is IL2.Another promotor p007 is that other parts are consistent with pf12 with the LTR of HIV-2 except second promotor.

What Fig. 9 showed is an amplification construct, and its therapeutic gene is IL2, by HIV-1 or HIV-II promoter regulation.CMV or the control of HSP70 promotor that the amplification part is expressed by regulation and control TAT.This plasmid also contains neomycin resistance gene and the necessary element of growth in bacterium.Such construct is used to the amplification analysis of example 2 and 3.Fig. 9 A comprises the plasmid X14 of CMV-TAT-HIV-1-IL2 expressed sequence.Fig. 9 B comprises the plasmid Y15 of CMV-TAT-HIV-2-IL2 expressed sequence.Fig. 9 C comprises the plasmid pf12 of HSP-TAT-HIV-1-IL2 expressed sequence.Fig. 9 D comprises the plasmid p007 of HSP-TAT-HIV-2-IL2 expressed sequence.

Figure 10 shows in the p173OR plasmid of StressGen Biotechnology Corp company and comprises the segmental dna sequence dna of BamH1-Hind III.This fragment comprises about 400bp minimum, HSP70B promoter fragment, and this fragment usually is used for special construct, as the following example that will mention 1 and example 3.

The narration of preferred embodiment

1. introduction of the present invention

Gene therapy faces two major technology difficult problems: how to regulate and control and strengthen therapeutic gene expression in vivo.The present invention has solved this two difficult problems by pyroprocessing and inducible expression's construct are integrated.The inventor is verified can to improve specific inducible expression's expression of gene efficient.

Expressing therapeutic gene high-levelly and effectively controlling expression level is developing two important topics of gene therapy.But the inventor has invented one group of new efficient expression vector overcomes this two difficult problems, and the inventor uses the amplification strategy to control the expression of goal gene.These amplify son is made up of people's HSP70B promotor, the expression of the trans activating transcription factor of this other promotor of promotor start-up control, and these trans transcription factors start the expression of reporter gene again conversely.These extra promotors are included in the reporter gene that links to each other with them to be had in HSP70B promotor element and the same carrier to the trans-activator coding.

In the mammalian cell transfection research of end user's IL-2 as reporter gene, the inventor shows, compare (face specific examples as follows with the reporter gene expression rate that starts separately by composing type CMV promotor or HSP70B, example 3), the amplification construct is in the temperature of all tests, and the expression of reporter gene all is improved largely.Contain the HSP70B promotor simultaneously, human immunodeficiency virus (HIV) upstream, tat gene and HIV1 or HIV2 long terminal repeat, the construct of interleukin-2 upstream region of gene has been showed under 37 ℃ of the specific activitys of promotor at high temperature to be improved largely.The test of mammalian cell cotransfection shows that promoter expression constructs HSP, the expression efficiency of HSP/HIV I and HSP/HIV2 are respectively 0.4,6.9,83.3 times that CMV expresses promotor.These data show: though the expression efficiency of minimum heat-inducible promoter is lower than CMV promotor, it can be united with second promotor and amplifies expression efficiency significantly, also preserves the dependence to temperature to a certain degree simultaneously.

Early stage research all is to start trans-activating factor with heat-inducible promoter to express, thereby activates other promotor (Schweinfest etc., Gene, 71 (1): 207-210,1988; EPO01 18393; WO 89/00603, United States Patent (USP) 5,614,381, United States Patent (USP) 5,646,010; EP 0 299 127).The present invention proposes and be different from early stage method, for example: 1) different heat-inducible promoter (Schweinfest etc. use the Drosophila promotor); 2) different transporting mode (the present invention is incorporated into two promotors in the simple construct-other method also using cotransfection); 3) different inducing temperature (temperature used of early stage research work is higher than 42 ℃, and the present invention can operate under 42 ℃ and lower temperature); 4) be applicable to gene therapy rather than industrial production.Also have, the expression level that the present invention can use HIV-1 or HIV-2 promotor and demonstrate both has notable difference.

A preferred aspect of the present invention is usually to control the expression of transgenosis in mammalian cell by being suitable for heat-inducible unit, the heat shock sequence is used for starting the trans-activating factor expression of gene, therefore when expression construct was given high temperature induction, the trans factor began to express.Trans-activating factor starts second promotor, and then starts the expression of therapeutic gene.In a special embodiment, also used the promotor of develop from the HSP70 promotor, a useful especially characteristic of this promotor is that its expression level under normal envrionment temperature is very low and be evoked promoter.The method that the present invention also provides the expression that makes therapeutic gene to reach the level of treatment requirement is come cell growth inhibiting or activating immune system.

Will go through composition relevant and method below with the present invention.

2. heat shock response

Heat shock or irritant reaction extensively are present in from plant to mammiferous tissue, it is not only the result of heat shock, and be that other comprises local asphyxia, anoxic, glucose lacks, ionization glucose and amino acid analogue, ethanol, transition are metal (transitionseries metals), medicine, hormone, the result that bacterium and virus infection etc. stimulate.The overexpression that also shows heat shock protein on evidence stimulates relevant with hyperplasia and tumour cell.(Finch etc., Cell Growth and Differentiation 3 (5): 269-278,1992).The principal character of heat shock response is a synthetic family sequence high conservative but protein that molecular weight does not wait, and these proteinic inductive conditions are different with locating.These protein are high conservative between kind, and they are divided according to its molecular weight.

When environmental change or health come to harm, the intensity of activation that protein gene is transcribed took place in several minutes, so fast speed of response lacks intron and makes heat shock protein can at high temperature walk around the sealing of intron to gene owing to the gene deletion intron of most of heat shock protein.Can make the heat shock protein high-efficient expression like this, through regular meeting other proteic expression rate be descended simultaneously.

The activation of stress gene is that the structure by thermal excited transcryption factor (HSF) changes mutually and regulates, and when being upset, the albumen that has existed is transformed into active structure phase mutually by nonactive structure.The species variation of the protein-bonded molecular weight of this DNA huge (for example, the people be 83kDa and yeast is 150kDa).The heat shock element is the conservative upstream regulatory sequence of HSP70, and HSF works with its combination.Though the major function of heat shock protein is to help protein folding and prevent protein coacervation, evidence suggests that they work in the aversion response of organizing stimulation to external world and wound recovery.

Similar with the Eukaryotic sequence specificity of major part transcription factor, the reactive element of HSF by the high conservative that exists with multiple copied with the heat shock gene upstream is in conjunction with working.The heat shock response element oppositely repeats 5 base sequences by three successive to be formed, and this base sequence is nGAAn, but more generally be AGAAn.The regulating effect of HSF mainly is active transformation rather than change synthetic and stability.

3. hyperthermia treatment

A lot of clinical studyes show that high temperature is effective (Hahn, G.M.Hyperthermia and Cancer, second edition, New York, Plenum, 1982 as radiotherapy and chemotherapeutical householder method to a lot of malignant diseases; Scott etc., Int.J.Rad.Oc.Biol, Phys.10 (11) 2219-2123,1984; Lindhol etc., Rec.Res in Cancer Res.107:152-156.1988).Heat shock is used, and adaptation and incompatible theory are to use on experimental evidence that can produce remarkable physiological action and the basis that contrasts clinical study in heat shock to grow up.Lehman has proposed about heat shock treatment (Therapeutic Heat and Cold, Rehabilitation Medicine Library, the Williams ﹠amp of proving in the application in other field; Wilkins publishes, and 1990, be incorporated herein this paper as a reference).The reader pays special attention to chapter 9, and the application of heat shock in the department of medicine and surgery treatment has been discussed therein.

" high temperature " typically refers to the temperature that is higher than test body envrionment temperature of living in.High-temperature temperature used herein is normally between 37 ℃ and 42 ℃.In some special embodiment, this temperature can in further embodiments, be 39 ℃ to 41 ℃ from 38 ℃ to 42 ℃, also has among some embodiment, and this temperature approximately is 40 ℃.Under at present the getable technology of institute helped, high temperature was used as assisting therapy, can make temperature remain on 42 ℃ differ in 0.5 ℃ the scope.Therefore, treatment of the present invention is handled can be at 37.0 ℃, and 37.2 ℃, 37.4 ℃, 37.6 ℃, 37.8 ℃, 38.2 ℃, 38.4 ℃, 38.6 ℃, 38.8 ℃, 39.2 ℃, 39.4 ℃, 39.6 ℃, 39.8 ℃, 40.2 ℃, 40.4 ℃, 40.6 ℃, 40.8 ℃, 41.2 ℃, 41.4 ℃, 41.6 ℃, 41.8 ℃, or 42 ℃.Before the present invention, effectively hyperthermia treatment requires temperature in the tumour to remain on more than 43 ℃ 30 to 60 minutes, and the safety temperature of healthy tissues is below 42 ℃, is very difficult and temperature is remained on more than 42 ℃, often is difficult to accomplish.

Can come cell in the heat shock Mammals with a series of technology, these technology comprise ultrasonic, electromagnetic technique, electromagnetic technique comprises keying wave (as microwave) again, resistance (as radiation) or inducibility (as radiation or hertzian wave) generator (Hahn, G.M_Hyperthermiaand Cancer, second edition, New York, Plenum, 1982; Lehman, L.B_Postgard Med_88 (3): 240-243,1990; Be incorporated herein this paper as a reference): in some simple application, tissue temperature can heat with round-robin hot water or warm air.

The United States Patent (USP) 4,230,129 (being incorporated herein this paper as a reference) of LeVeen, this article mention a kind of under the help of scintillation counter heatable body tissue and detect the method that the tumour cell internal temperature changes.This method is to make radiation (RF) energy and body-coupled to avoid the excessive absorption radioactivity of fatty tissue heat, and making the radioactivity energy by a kind of therapeutic device do orbiting is not to concentrate on the intravital a certain zone of body to concentrate on tumour cell.The United States Patent (USP) 4 of LeVeen, 230,129 (being incorporated herein this paper as a reference), mentioning a kind of method in this article is to place the human body parts that has tumour cell a radioactivity electromagnetism zone to heat tumour cell, and does not injure the healthy tissues that links to each other with tumour cell.

In the embodiment that the present invention uses always, pyroprocessing and gene therapy vector are united use, induce the expression that has changed the tumor locus therapeutic gene over to high temperature.And high temperature/gene therapy treatment process also can unite use with other traditional methods of treatment such as chemotherapy to be discussed below and beamtherapy etc., to obtain better effect.Other high temperature induction methods of treatment is seen technology chapter.See correlation technique brief introduction chapters and sections about the zone of hyperthermia treatment method or system application method and instrument, also can in following american documentation literature, find relevant narration simultaneously: United States Patent (USP) 5,284,144; 4,230,129; 4,186,729; 4,346,716; 4,848,362; 4,815,479; 4,632,128, all be incorporated herein by reference at this.

4. quantity sheet expression constructs

In some special embodiment, present invention includes the method for genetic material being operated the expression construct that obtains cloning therapeutic gene.The step of these methods comprises: use containing the carrier of the proteic foreign DNA of coding treatment and the approach that its is expressed, and duplicates this carrier then with the acquisition virion in helper, and then removes cells infected with the virus of reorganization.This foreign gene generally is a therapeutic gene, as the gene of kill cancer cell, and the gene of defective in the immunomodulatory gene of anti-virus infection or the replacement body.In virus vector, therapeutic gene is an external source, that is to say that it is not from the viral genome that constitutes carrier framework.At last, virus can be used as vaccine alive and antigen expressed to stimulate the expression of specific antibodies.Foreign gene can be from protokaryon or eukaryote, as bacterium, and virus, yeast, parasite, plant or animal, also can be from a more than approach such as polyclone gene construct or a fusion rotein, foreign DNA also can comprise one section with self different adjusting sequence in source.A) therapeutic gene

Selected poly-nucleotide among the present invention can be used as therapeutic gene and plays a role.The therapeutic gene of any wide material sources all is applicable to carrier and the method among the present invention.The present invention can be used for therapeutic gene special pathology, malpractice, or treatment of diseases related to the present invention.

In one embodiment of the invention, specific poly-nucleotide is coding ornithine decarboxylation plum (ODC) antizyme, this enzyme is an important feedback regulation factor (Hayashi etc., Trends in Biochemical Sciences 21 (1): 27-30,1996) of polyamines in the cell.This proteic expression level is directly related with intracellular polyamine level, and it can stimulate this proteic expression.Antizyme acts on ornithine decarboxylation plum, and it is polyamines first enzyme in synthetic, often also is the rate-limiting enzyme polyamines of degrading.This albumen also suppresses the absorption of polyamines.Therefore low-level polyamines causes low-level antizyme to be expressed, and low-level antizyme causes the synthetic of and the polyamines highest level taken in synthetic by ODC.On the contrary, high-caliber polyamines causes high-caliber antizyme to be expressed, and high-caliber antizyme makes polyamines synthetic with minimum level.

Radio-protector WR-33278 (N, N " (dithiodi-2; 1-ethanediyl) bis-1; 3-propanediamine) contain many ammonia analogue of two sulfonium keys; cell can by many ammonia transhipment absorb it (Mitchell etc., Carcinogenesis, 16:3063-3068; 1995), this transhipment is sub to be suppressed by antienzyme.The WR-33278 animal test model shows that some healthy tissues can absorb more WR-33278 (Ito etc. than some tumours at least; InternationalJournal of Radiation Oncology; Biology; Physics 28:899-903; 1994) reagent resemble WR-33278 has been applied to clinical radiation treatment, and it can be protected the healthy tissues of dose limitation and not reduce simultaneously the effect of radiation therapy to tumour cell.(Spencer and Goa, Drugs, 50 (6): 1001-31,1995, be incorporated herein this paper as a reference).The difference theory that absorbs about WR-33278 can be summed up as the non-hyperplasia normal cell of outgrowth tumor cell ratio and contain more many ammonia, so tumour cell is than the higher levels of antienzyme of normal tissue expression.

The present invention places the antienzyme cDNA that has lacked the necessary sequence of many ammonia dependency regulation and control under the regulation and control of people's heat-inducible promoter HSP70B, and the present invention transforms by the develop DU-145 clone of coming and to have filtered out the clone who has the many ammonia of heat-inducible to absorb be (show thermal induction antienzyme activity is arranged) of Human Prostate Cancer Cells with this construct.This gene therapy method (expression of HSP70B promoter regulation antienzyme) can be used for the clinical trial of patients with prostate cancer in the future, and this method can and be located radiation combination therapy prostate gland patient with WR-33278.When the antienzyme in the tumour cell is positioned the activated at expression, thereby the normal dose limitation cell adjacent with cancer cells can at high temperature do not expressed antizyme and be absorbed antiradiation agent WR-33278, and tumour cell can not absorb antiradiation agent owing at high temperature express antizyme simultaneously.Thereby the radiation irradiation that this method allows prostatic to be carried out high dosage kills prostate tumor cells.

In another embodiment; cell-protecting medicines ethol (also is amifostine; WR-2721; or S-2-(3-aminopropylamino) ethylphosphorothioic acid) other meta-bolites also can be united use with the expression construct of introducing herein; for example, WR-1065 (2-(3-aminopropylamino) sulfur alcohol) also can be used as radiation protective.

Also have other a lot of gene also can transform with carrier of the present invention, for example, carrier among evidence the present invention can be used for changing over to tumor suppression, the prodrug of the former dust gene of antisense and picture HSV-TK gene and so on activates son (Rosenfeld etc., Annals of Surgery, 225:609-618,1997; Esandi etc., Gene Therapy, 4:280-287,1997) wait and treat cancer.Other gene that can utilize expression construct of the present invention to change over to comprises p53, p16, p21, p27, C-CAM, HLA-B7 (Gleich etc., Arch Otolaryngol HeadNeck Surg, 124:1097-104,1998; Heo etc., Hum.Gene Ther.9:2031-8,1998; Nabel etc., Proc.Nat.Acad.Sciences USA, 90:15388-15393,1996; Stopeck etc., Journal of Clinical Oncology, 15:341-349,1997), IL2 (O ' Malley etc., Molecular Endocrinology, 11:667-673,1997; Otova etc., Folia Biologica, 43:25-32,1997), IL4 (Kling, Nature Biotechnology, 15:316-317,1997), IL7 (Toloza etc., Annals of Surgical Oncology, 4:70-70,1997); Sharma etc., Cancer Gene Therapy, 3:303-313,1996), IL12 (Hiscox and Jiang, InVivo, 11:125-137,1997; Chen etc., Journal ofImmunology, 159:351-359,1997), GM-CSF (Kreitman and Pastan, Blood, 90:252-259,1997; Homick etc., Blood, 89:4437-4447,1997; Lanza etc., Haematologica, 82:239-245,1997), IFN γ (Noguchi etc., Mclinical Infectious Diseases, 24:992-994,1997; Kanemaru etc., European Archibes of Oto-Rhino-Laryngology, 254:158-162,1997; Tanaka etc., Journal of Gastroenterology and Hepatology, 11:1155-1160,1996; Imai etc., Liver, 17:88-92,1997), I-CAMI and TNF (Corcione etc., Annals of the New York Academy of Sciences, 815:364-366,1997).(all be incorporated herein by reference at this.)

P53 be it is believed that it is a tumor suppressor gene (Montenarh, Crit.Rev.Oncogen, 3:233-256,1992) recently.By chemical carcinogen, found to have high-caliber sudden change p53 to express many in the malignant virus cell transformed of uviolizing and SV40.The P53 gene is the sudden change inactivation in a lot of people's tumours, and it also is the gene of easy sudden change in the human cancer, and it also suddenlys change in surpassing 50% human NSCLC and a lot of other tumours.

P16 ^INK4Belong to a new CDK-arrestin family, this protein family comprises p16B, p21 ^{The WAF I, CIP I, SD II}And p ^27KIP1p ^16INK4Gene is positioned at chromosomal 9p21 zone, and this zone is deleted in a lot of tumour cells.P16 ^TNK4The homology deletion and the sudden change of gene are very general in a lot of human tumor cell lines, and it shows that this gene is a tumor-inhibiting factor.Yet this inference along with the change probability that it is found that this gene in the non-culture of tumor cell of primary more than will be low in the culture of tumor cell system and change.With containing wild-type p16 ^INK4Can reduce transformed cell cloning in some cancer cells of plasmid expression vector transfection of gene.(Okamoti etc., Proc, Natl, Acad.Sci, USA, 91:11045-11049,1994:Arap etc., Cancer Res_55:1351-1354,1995, all be incorporated herein by reference at this).

C-CAM is effective expression in all epithelial cells, and its molecular weight is approximately 105kD, is at first to come out with the specific antibodies of anti-hemocyte cohesion purifying from the rat hepatocytes plasma membrane.Nearest studies show that structurally C-CAM belongs to immunoglobulin (Ig) (Ig) superfamily, and its sequence and carcinomebryonic antigen (CEA) are the height homologous, and first Ig structural domain of C-CAM is necessary to its cell adhesion activity.

Cell adhesion molecule (CAM) has participated in regulating the complicated molecule effect network (Edelman, Annu.Rev.Biochem_54:135-169,1985) of tissue development and cytodifferentiation.Nearest testing data shows that the unconventionality expression of CAM is relevant with the generation of malignant tumour, and for example E-cadherin (remarkable expression is arranged in the epithelial cell) reduction of expression level is relevant with the generation of some tumour.Giancotti and Ruoslahti (Giancotti and Ruoslahti, Cell, 60:849-859,1990) prove external and change the tumour generation that the expression that improves integral protein α 5 β 1 can reduce Chinese hamster ovary cell over to by gene.C-CAM can suppress the growth of tumour cell in vitro and in vivo.

Other tumor-inhibiting factor also can be used in the present invention.For example, selected poly-nucleotide can be any one in the following gene: retinoblastoma gene (Rb); Adenoma porous colon gene (APC); The colorectal carcinoma gene (DCC) of defective; Neurofibromatosis gene 1 (NF-1); Neurofibromatosis gene 2 (NF-2); The tumor suppressor gene of Wilm (WT-1); One many endocrine tumorses of type gene (MEN-1); Two many endocrine tumorses of type genes (MEN-2); BRCA1; Von Hippel-Lindau syndromes gene (VHL); Disappearance colorectal carcinoma gene (DCC); P16; P21; P57; P27 and BRCA2.

In another embodiment of the present invention, it provides method and the carrier that starts regenerative process (as neurotization) by the stimulating growth factor and cytokine expression.In this embodiment, specific nucleic acid can be a neural factor.The ciliary neurotrophic factor of for example can encoding (CNTF), the neural factor (BDNF) that the brain evolution comes, neural factor (GDNF) (Mitsumoto etc., Science, 265:1107-1110,1994 that the glial cell line evolution comes; Cash etc., Ann.Neurol_44 (3 suppl 1): s121-125,1998, all be incorporated herein by reference at this).Specific nucleic acid in the expression vector tyrosine hydroxylase of also can encoding, GTP cyclization hydrolase 1, aromaticity L-amino acid decarboxylase plum (Kang, Mov, Disord_13 supp 11:59-72,1998, all be incorporated herein by reference at this).In another embodiment, the framework body is expressed in treatment can express a somatomedin, as insulin-like growth factor 1 (IGF-1) (Webster, Mult.Scler_3:113-120 1997, all are incorporated herein by reference at this).

The present invention not only can be used for the treatment of the disease and the pathology of high hyperplasia type, can also be used for other as rheumatoid arthritis or (after referring to cardiac valve procedure especially) restenosis treatment of diseases such as (renstenosis) by the therapeutic gene that changes over to as coding blood vessel generation supressor or cell cycle supressor.B) antisense constructs

Such as ras, myc, neu, raf, erb, sec, fins, jun, trk, ret, gsp, hst, proto-oncogenes such as bcl and abl are suitable therapeutic genes.Yet for the result of treatment that obtains, these proto-oncogenes are the formal representation with antisense nucleic acid expression of suppressing proto-oncogene." antisense nucleic acid " is meant and proto-oncogene coded DNA or RNA complementary nucleotide sequence, when antisense nucleic acid is expressed in target cell, it is specific combines with target nucleic acid sequence that interference is transcribed, RNA synthetic, transhipment or translation, when it forms the triple helical structure mutually with the double-stranded DNA bonded time, double-stranded structure mutually in the time of with the RNA bonded.

Antisense constructs can be designed to promotor and other regulation and control zone with a gene, exon, and intron, even exon-intron joining region bonded form is sealed expression of gene.No matter external or comprise that people's in vivo test, the DNA construct of sense-rna construct or coding RNA S can be used for suppressing transcribing of host cell or translation or both and suppress.The nucleotide sequence that comprises complementary nucleic acid be can be under the Wstson-Crick of standard principle paired.This principle is that big slightly purine and the pairing of slightly little pyrimidine form G:C and A:T matching form in DNA, form the matching form of A:U in RNA.

" complementary sequence " mentioned herein or " antisense sequences " are meant that nucleotide sequence is to stablize complementary on whole sequence, and mispairing is seldom arranged.For example, if one section nucleotide sequence that 15 bases are arranged it have 13 or 14 (one or two mispairing) bases and the pairing of another section sequence just can be called complementation.Generally, the nucleotide sequence of " fully complementary " is complementary fully and do not have a mispairing on entire segment.

All or portion gene sequence can be used for antisense constructs, and statistics shows that the long sequence of any one section 17 base only can occur once in genome, and this is enough to guarantee that it has only a unique target sequence.Though short nucleic acid sequences prepares easily and changes over to easily in vivo, also has its hybridization specificity of other a lot of factor affecting.By being 8,9 to length, 1O, 11,12,13,14,15,16,17,18,19,20 or more the nucleotide sequence of polybase base be used to analysis of experiments, the increasing with its length of nucleic acid in conjunction with specificity and sequence specificity.Whether the Expression of Related Genes that whether influences corresponding native gene function or contain complementary sequence by analytical table expression constructs in the in vitro tests is affected, and we can be easy to judge whether antisense nucleic acid can work in target cell.

In some specific embodiments, people wish to comprise in the antisense constructs other element, for example C-5 propyl group pyrimidine.The poly-nucleotide that evidence contains the C-5 propyl group analogue of uridylic and cytosine(Cyt) can combine with the high affinity of RNA, be potential genetic expression antisense inhibitor (Wagner etc., Science, a 260:1510-1513,1993, be incorporated herein this paper as a reference).C) ribozyme construct

Ribozyme can be used for substituting antisense constructs." ribozyme " is to discern and to cut the normally RNA molecule of the specific base sequence in the RNA molecule of NDA.In the present invention, ribozyme is imported into cell and ribozyme expression RNA in cell.The target sequence of ribozyme is identical with the target sequence of antisense nucleic acid.

A lot of ribozymes can be in the hydrolysis of catalysis phosphodiester bond under the physiological condition.Ribozyme energy catalysis among the present invention is to the particular sequence cutting of another nucleic acid molecule, and normally one section mRNA molecule of transcribing also can be the mRNA that proto-oncogene is transcribed.Generally ribozyme is by being positioned at its enzyme lateral binding site in site alive and target RNA combination, and RNA is cut in enzyme site alive then.Can destroy the ability of its proteins encoded directly or indirectly to the deletion of target RNA.After the ribozyme cutting was finished, it discharged and seeks next target body from target RNA.

In a preferred embodiment of the invention, the ribozyme of application is a brain malleus ribozyme, and it is microRNA (Symons, Ann.Rev.Biochem.61:641-671,1992 come from the plant virus evolution; Clouet-D ' Orval and Uhlenbeck, RNA, 2:483-491,1996; Haseloff and Gerlach, Nature 334:585-591,1988; Jeffries and Symons, Necleic Acids Res, 17:1371-1377,1989; Uhlenbeck, Nature, 328:596-600,1987; All be incorporated herein by reference at this).

In other embodiments, ribozyme can be first group of intron, hair fastener ribozyme, VSRNA, hepatitis Delta virus ribozymal or Rnase P-RNA ribozyme (relevant with one section RNA homing sequence).See article Hampel etc. about the effect of hair fastener, Nuclei Acids Res.18:299,1990 and Hampel and Tritz, Biochemistry 28:4929,1989; Hepatitis delta virus is seen the article of Perrotta and Been, Biochemistry 31:16,1992; The United States Patent (USP) 5,624,824 of Yuan etc. has been described the example of RNAsep; Article at Sabulle and Collins, Cell 61:685-696,1990, the article of Saville and Collins, Proc.Natl.Acid.Sci.US A 88:8826-8830,1991, the article of Collins and Olive, Biochemsitry 32:2795-2799 (1993) has described neurospora VS ribozyme rna.The article of Ceth etc., United States Patent (USP) 5,354,855 are seen in the description of first group of intron.The present invention not only is confined to ribozyme above-mentioned, the ribozyme that contains with said target mrna complementary substrate binding site all can be applied among the present invention, the enzyme that this ribozyme also contains the cutting RNA molecule site of living, it is positioned at the inside of substrate binding site or around it.D) selected marker

In certain embodiments of the present invention, no matter in vivo or external, the expression of the therapeutic gene in the expression vector can be identified by a marker of carrier inside.Like this some marks can make the cell generation noticeable change that changed this expression vector over to and easily and other cell separate.Usually in expression construct and selection transformant, all contain a medicament selection mark.For example: can use anti-new certain kind of berries element in the expression vector, purine certain kind of berries element, Totomycin, DHFR, GPT, the gene of zeocin and histidinol is as selective marker.Certainly resemble that simple luxuriant exanthema virus chest pyrimidine swashs enzyme such as plum and immunoglobulin (Ig) also can be used as selection markers.As long as express simultaneously with therapeutic gene, select which type of selective marker just to seem very unimportant so.Other more selected marker comprises EGFP, and β-gal becomes transferring enzyme (CAT) with the plain second of the chlorine certain kind of berries.E) complex gene construct and internal ribosome binding site (IRES)

In specific embodiments more of the present invention, used internal ribosome binding site (IRES) unit and usually produced complex gene or polycistronic messenger.The IRES element can make rrna the time break away from 5 in translation '-dependence of the cap that methylates and internally the site begin translation.IRES element (Pelletier and Sonenber from two members (polio and encephalomyocarditis) of virus family picanovirus, Nature, 334:320-325,1988) with from IRES (Macejak and Sarnow in the Mammals courier gene, Nature, 353:90-94,1991) clear by elaboration.The IRES element can be connected with the open reading frame of heterologous gene.Multiple reading frame can insert the IRES element by the centre and connect together and transcribe the generation polycistronic messenger.Under the help of IRES element, each reading frame can effectively be translated with the rrna combination, so just can transcribe complex gene by a simple promotor/enhanser, produces single courier.

The open reading frame of any external source can be connected use with the IRES element.These genes comprise the gene of the secretory protein of encoding, multimeric protein, intracellular protein or the symphysis albumen and the selectable marker gene of separate gene coding.F) regulation and control zone

In order to make the transcriptional expression of the therapeutic gene in the control expression vector, must place it under the regulation and control of promotor and polyadenylic acid signal." promotor " is meant can be by the dna sequence dna of the synthesis system of the synthesis system of host cell or importing identification, and it is necessary that it is that promotor gene is transcribed.The polyadenylic acid signal is meant can be by the dna sequence dna of the synthesis system of synthesis system in the host cell or importing identification, it to mRNA transcribe when finishing to it add a series of nucleic acid be essential with mediated mRNA from examining cytoplasmic transfer." transcriptional control " is meant that promotor is positioned at the accurately site relevant with specific nucleic acid, and the initial sum of rna regulation polysaccharase is expressed.

Promotor is meant that here is positioned at a RNA polymerase II initiation site transcriptional control molecule on every side.Promotor is that a lot of theories of this problem how to organize are to draw by the analysis and research to several viral promotors when transcribing, comprising the early transcription unit of HSV chest pyrimidine kinases (tk) and SV40.These researchs (recently more work increase its demonstration) show that promotor is molecular by some discontinuous functions, and wherein each function contains about 7-20 base, one or more activating transcription factors or suppress sub-binding site.

The function that has function in each promotor at least is to determine RNA synthetic initiation site.Foremost example is exactly TATA box, but there are some promotors to lack TATA box, as the late gene promotor of Mammals terminal deoxynucleotidyl transferase and SV40, TATA box is a discontinuous element that self covers initiation site, the transcription initiation site of its decision RNA polymerase.

Other promotor element is regulated the frequency of transcripting starting.These promotor elements are positioned at the zone of initiation site upstream 30-110bp generally speaking, but nearest a lot of promotors that studies show that contain the functional element that is positioned at the initiation site downstream.Sequence between the promotor element is flexible and variable, does not therefore influence the function of promotor when these elements are inverted or change relative position.In the tk promotor, the distance when between the promotor element increases that to obtain 50bp be that promotor active just begins decline.As if in promotor, the promotor element can be worked in coordination with or independent activated transcription.

When target cell is human body cell, at first will the coding region of therapeutic gene with can be in human body cell expression promoter be connected and under its regulation and control.Generally, this class promotor comprises the promotor of people or virus.Table 1 is some promotor tabulations.

Table 1

Promotor
	Heavy chain immunoglobulin
Light chain immunoglobulin
	T-cell receptors
HLA DQ α and DQ β
	Beta-interferon
Interleukin II
	Interleukin
2 receptor
	????MHC?ClassⅡ5
????MHC?ClassⅡHLA-DRα
	β-motor-driven albumen
The acid of muscle machine swashs plum
	Prealbumin (Transthyretin)
The elastoser I
	Metallothionein(MT)
Collagenase

(being contiued from the previous page)

Albumin gene
	α-fetoprotein
τ-globin
	Beta-globin
????c-fos???
	????c-HA-ras
Regular Insulin
	Nerve cell adhesion molecule (NCAM)
Alpha1-antitrypsin
	H2B (TH2B) histone
Mouse or type I collagen
	Glucose regulated protein (GRP94 and GRP78)
Rat growth hormone
	Human serum amyloid (SAA)
Troponin I (TNI)
	The little version of blood deutero-somatomedin
The tubular muscle malnutrition
	????SV40
Polyoma (Polyma)
	Retrovirus
Nipple virus
	Hepatitis B virus
Human immunodeficiency virus
	Cytomegalovirus
Gibbon Ape leukosis virus

To the requirement that is used to control the special promoter that therapeutic gene expresses is not very fastidious, just can as long as it can be activated by the gene product that evoked promoter is controlled.In a preferred embodiment of the invention, trans-activator is tat, and the promotor that links to each other with therapeutic gene is the long terminal repeat of HIV-1 or HIV-2.For example, the promotor element that contains the AP-1 site can be subjected to the c-jun or the proteic regulation and control of c-fos of abduction delivering, and other trans-activating factor/promotor integrated mode that is fit to sees for details in this technology.

The promotor of control trans-activating factor genetic expression must be an evoked promoter.Evoked promoter does not have activity or active relatively low under the situation of not inducing substrate to exist.The promotor of using among the present invention includes but is not limited to MTII, the MMTV collagenase, and stromelysin, SV40, mouse MX gene, α-2-macroglobulin, I type mhc gene h-2kb, increment albumen, tumour necrosis factor, Triiodothyronine stimulates the α gene.The stimulating factor of this gene-correlation sees Table 2.Egr-1 promotor and multiple drug resistant gene (MDR1) promotor also are suitable evoked promoters.Usually evoked promoter is a heat-inducible under the situation, and it is that from following promotor one develops: HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin and HSP28.In another kind embodiment commonly used, evoked promoter comprises a hypoxemia reactive element, as HIF-1.What should understand is that any one evoked promoter can be used in the present invention, and all such promotors do not exceed core concept of the present invention and scope.

Table 2

The promotor element	Inductor
		????MTII	Negative ion fat (TPA), heavy metal
MMTV (mouse corpora mammillaria tumour virus)	Glucocorticosteroid
		Beta-interferon	????Poly(rl)X,poly(rc)
Adenovirus 5E2	????Ela
		????C-jun	Negative ion ester (TPA), H 2O 2
Collagenase	Negative ion ester (TPA)
		????Stromelysin	Negative ion ester (TPA), IL-1

(being contiued from the previous page)

????SV40	Negative ion ester (TPA)
		Mouse MX gene	Interferon, rabbit, Avian pneumo-encephalitis virus
The GRR78 gene	????A23187
		α-2 macroglobulin	????IL-6
????Vimentin	Serum
		I type mhc gene H-2kB	Interferon, rabbit
????HSP70	Ela, the SV40 large T antigen
		Proliferin (Proliferin)	Negative ion fat-TPa
Tumour necrosis factor	????FMA
		Sugar cortex stimulates hormone α gene	Glucocorticosteroid

In some specific embodiments, trans-activating factor is a tat albumen.The genome of the genome of HIV-1 and HIV-2 virus and simian immunodeficiency disappearance virus (SIVS) has very high homology, all they has been carried out deep research.Discover except gag, env, pol gene in all upset record virus be identical beyond, it also is identical also having many regulatory gene that play an important role transcribing of virus.The viral protein tat regulatory factor that comes to this, it can improve the activity of HIV-1 and HIV-2 promotor significantly.(Sodroski etc., J.Virol_55 (3): 831-835,1985a; Sodroski etc., Science, 229 (4708): 74-77,1985b; Sodroski etc., Science, 228 (4706): 1430-1434,1985c; Sodroski etc., Science, 227 (4683): 171-173,1985d; All be incorporated herein by reference at this).Tat albumen be considered to HIV LTR in trans-activation element (TAR) in conjunction with and improve the specific rna level of stable HIV.Evidence suggests that also tat not only works as other traditional trans-activating factor, it can also interact with other trans-activating factor.Tat and adenovirus trans-activating factor ELA can improve the level of stablizing RNA significantly.(Laspis etc., Genes Dev_4 (12B): 2397-2408,1990).Like this, one can further to improve the active method of HIV-LTR/TAT construct be that EIA is incorporated in this construct.

In the place that construct inserts cDNA, to contain a polyadenylic acid signaling zone usually so that can control the normal polyadenylic acidization of open gene.In practical application of the present invention, the characteristic of polyadenylic acid signaling zone is unimportant, and any such sequence may be used among the present invention.Look like from SV40, Trobest, the polyadenylic acid signaling zone of simple luxuriant exanthema virus chest pyrimidine kinase gene has been successfully applied in a lot of cells.

5. gene changes mode over to

Control the transit cell expression of gene, at first will import the therapeutic gene expression construct among the present invention or change in the cell, such mode that changes over to can be a virus or non-virus-mediated.This part will be discussed the method and composition that gene changes over to.A. non-virus changes mode over to

In a preferred embodiment of the invention, different genetic expression construct must change cell over to and just can work.In some cases, changing over to by non-viral form of expression construct undertaken.The invention provides several non-virus-mediated transfer methods that expression construct changed over to mammalian cell.These methods comprise calcium phosphate precipitation method (Graham and Van Der Eb, Vriology, 52:456-467,1973; Chen and Okayama, Mol.Cell.Biol_7:2745-2752,1987; Reppe etc., Mol.CellBiol_10:689-695,1990) DEAE-dextran method (Gopal, Mol.Cell.Biol, 5:1188-1190,1985), electroporation (Tur-Kaspa etc., Mol.Cell.Biol_6:716-718,1986; Potter etc., Proc.Nat.Acad.Sci.USA.81:7161-7165,1984), direct microinjection (Harlandand Weintraub, Mol.Cell.Bio1_101:1094-1099,1985), DNA filled liposome method (Nicolau and Sene, Biochim.Biophys.Acta, 721:185-190,1982; Fraley etc., Proc, Natl.Acad.Sci.USA, 76:3348-3352,1979), cell sonication method (Fechheimer etc., Proc.Natl.Acad.Sci.USA, 84:8461-8467,1987), high speed gene bullet method (Yang etc., Proc, Natl.Acad.Sci.USA, 87:9568-9572,1990), receptor-mediated transfer method (Wu and Wu, J.Biol.Chem_262:4429-4432,1987; Wu and Wu, Biochemistry, 27:887-892,1988, above document chapters and sections all are incorporated herein this paper as a reference).

In case the coding therapeutic gene changes cell over to, it can be located in different sites and express.In some cases, therapeutic gene can successfully be incorporated in the cellular genome.These integration sites can be homologous site and homologous recombination (gene replacement) initiation site, also can be at random, nonspecific site.Yet the nucleic acid that changes in certain embodiments, can with independently, free dna fragmentation stable existence is in cell.These nucleic acid fragments or " free son " contain is independent of oneself or the application cell synthetic system keeps and duplicate oneself information.How expression construct being changed over to cell and it is positioned at cell interior and where depends on expression construct.

In specific embodiments more of the present invention, expression construct can be embedded in the liposome and change over to.Liposome is the balloon-shaped structure body, and its feature is the aqueous media that contains double-deck immobilized artificial membrane and film inside.Multilamellar liposome separates the greasiness layer by water and forms, and is that it can spontaneous formation when phosphatide is suspended in the excessive aqueous solution.The fat composition oneself of elder generation before forming closing structure arrange, and then water between the double resin layer and solute embedding got up to form liposome.When joining DNA in the cationic-liposome, liposome can compress spherical structural transformation to the mobile fat body-crystal with opticity, and this DNA-liposome complex is a kind of potential non-viral gene treatment carrier.

Liposome-mediated nucleic acid transforms and the in vitro tests of the expression of foreign DNA in cell is very successful.People such as Wong (Wong etc., Gene, 10:87-94,1980, be incorporated herein this paper as a reference) prove the chick-embryo cell of liposome-mediated foreign gene by using beta-galactosidase enzymes as reporter gene in vitro culture, the Hela cell, conversion in the liver cell and expression are feasible easily.People such as Nicolau (Methods Enzymol, 149:157-176,1987, enumerate in the document) by injection in the body, having finished of success changes liposome embedded gene over to mouse.Some business methods that relate to " fat transfection " technology have also been comprised among the present invention.

In some specific embodiment of the present invention, liposome can mix use with hemagglmination polyprotein virus (HVJ), and it is easier to make the fusion of mixture and cytolemma and liposome embedded DNA enter cell like this.In some other embodiment, liposome can with endonuclear nonhistones-chromosomin (HMG-1) unites use.Also have among some embodiment, liposome can use jointly with HVJ and HMG-1.No matter these methods can both successfully change foreign gene over to and express in cell in vivo or in the in vitro tests, all are feasible for the purpose of the present invention therefore.

Other carrier transport system that can be used for therapeutic gene is changed over to cell comprises receptor-mediated delivery system.It is in nearly all eukaryotic cell that this method is utilized principle, is subjected to physical efficiency mediation selectivity to absorb macromolecular endocytosis.Because acceptor is to have the cell specificity, so transfer method has very high specificity (Wuand Wu, Adv.Drug.Delivery Rev_12; 159-167,1999).

Receptor-mediated transgenosis launch vehicle comprises two components: cell receptor-specific part and DNA binding substances.Several parts have been applied to receptor-mediated gene transformation.Most widely used part comprises asialoorosomucoid (ASOR) (Wu and Wu, J.Biol.Chem_262:4429-4432,1987) and transhipment (Wagner etc., Proc.Natl.Acad.Sci.87 (9): 3410-3414,1990).Recently, an artificial synthetic neoglycoprotein, its identification ASOR acceptor is used to gene and changes launch vehicle (Ferkol etc., FASEB J_7:1081-1091,1993 over to; Perals etc., Proc, Natl.Acad.Sci.USA, 91:4086-4090,1994) and Urogastron (EGF) also be used to change gene in the flakey tumour cell (Myers, EPO 0273085).

In other embodiments, the transhipment instrument can be made up of part and liposome.For example people (Methods Enzymal_149:157-176,1987) such as Nicolau is with the lactose ceramide, and semi-lactosi end-asialganglisside is embedded in transformant in the liposome, can observe liver cell and absorb the insulin gene level and improve.Like this, under the help that is with or without liposome, can be easy to specifically change therapeutic gene cells such as over to prostate gland, upper epidermis or tumour by the receptor-ligand system.For example: the specific antigen (Watt etc., Proc.Natl.Acad.Sci_83 (2): 3166-3170,1986) of human prostate can be used in the receptor-mediated system therapeutic gene is changed in the prostata tissue.

In another embodiment of the present invention, expression vector only is made up of naked recombinant DNA or plasmid.The conversion of this expression vector can be by any method of passing cytolemma with physics or chemical process above-mentioned.This carrier is particularly suitable for vitro conversion, can certainly be applied in the body.People such as Dubensky (Proc.Nat.Acad.Sci.USA, 81:7529-7533,1984) successfully with many property tumor virus DNA with the form of calcium phosphate precipitation be expelled to grow up or the liver and splenocyte of newborn mice in, observed that challenge virus duplicates and acute infection.Benvenisty and Neshif (Proc, Natl.Acad.Sci.USA, 83:9551-9555,1986) have proved that also the plasmid at the subcutaneous injection calcium phosphate precipitation can cause its expression in cell.Also there is gene that test shows the CAM that encodes also can change in the body and express CAM by similar methods.

The method that among the present invention the naked DNA expression vector is changed in the cell comprises gene bullet particle.This method be bag by the miniature bullet particle of DNA accelerate to a very high speed, make it pass cytolemma and enter cell but cell killing (327:70-73 1987, is incorporated herein this paper as a reference for Klein etc., Nature) not.Developed the method for several these minitype particles of acceleration.Wherein a kind of method is to rely on high-voltage discharge to produce electric current, and electric current produces miniature bullet that thrust (Yang etc., Proc, Natl.Acad.Sci.USA, 87:9568-9572,1990) uses conversely and is made up of biologically inert material such as tungsten or gold bead.B. the conversion of virus vector mediation

The another kind of method that realizes gene transformation is by with the gene transfer of the infective virus particle of tool as delivery means, for example, among the present invention following described be the conversion of carrier with the adenovirus.The retrovirus bovine papilloma virus also can be used as the alternate carrier, and the two all can for good and all change goal gene in the host cell over to.Therefore, in an example, the cell of virus infection is used to the effective gene of treatment usefulness is changed in the cell.Typically, virus is exposed to suitable cell simply under physiological condition, makes cell introduce virus.Though this sentences adenovirus as an example, present method can help to use on other virus vector, as described below equally.The those skilled in the art in field of the present invention are familiar with these methods very much.A) adenovirus

Adenovirus is because its medium sized genome, easy handling, and high-titer, broad host range, and high infectious, be particularly suitable for as the gene transformation carrier.Its total length 36kB viral genome two ends are the border with the long reverse terminal repeat (ITR) of 100-200 base (bp), wherein contain viral dna replication and the necessary cis acting element of parcel.Press the viral dna replication pattern, contained different transcription units are divided into (E) and (L) gene regions in late period in early days in the genome.

E1 district (E1A and E1B) encoded protein is responsible for the regulation and control that virogene and some cytogenes are transcribed.The expression in E2 (E2A and E2B) district produces the virus replication desirable proteins.Comprise viral dna replication, the sealing of late gene expression and host cell (Renan, 1990).The product of late gene (L1, L2, L3, L4 and L5) comprises main virus capsid protein, only expresses in effective processing back of a single primary transcription that is caused by late gene promotor (MLP).MLP (be positioned at 16.8 chromosome maps apart from) is active especially at later period of infection, and all are had terminal sequence (TL) before the trifolium of one 5 ' end by its mRNA that draws, it preferentially is translated.

In order to make adenovirus be suitable for gene therapy, need make it have maximum lift-launch capacity so that the dna fragmentation of parcel maximum.And very be necessary to lower toxicity and the immune response that some adenovirus product is brought.These two purposes all can be realized by the corresponding adenoviral gene of deletion.In actually operating of the present invention, can reach these purposes and make the treatment construct keep corresponding simple operation simultaneously.

It is possible that a large amount of DNA replaces, and the inverted repeats (ITR) that the necessary cis element of virus replication all is arranged in linear viral genome two ends (100-200).The plasmid that contains ITR can duplicate in the presence of the adenovirus of inactivation not.Therefore, containing this element in adenovirus carrier makes and duplicates and can realize.

In addition, Bing Du packaging signal is positioned at viral genome left end 194-385 bp (0.5-1.1 map distance) and locates.This class signal is similar to the albumen recognition site in the lambda bacteriophage dna, and one near left-end point but be positioned at special sequence outside the sticky end sequence, and mediation is attached on the necessary albumen in the DNA insert head structure.The adenovirus of E1 disappearance has shown that one 450 bp (0-1.25 map distance) that is arranged in the viral genome left-end point can guide the packing at 293 cells.

Some district that has before shown the adenoviral gene group can be incorporated in the mammalian cell and express genes carried.These clones can make the adenovirus carrier of these gene knock-outs be duplicated.The adenovirus carrier that also has report to show to duplicate inactivation can help carrier, causes the mutant of change as wild-type virus or condition, obtains the complementary replication under existing.

The adenovirus carrier of replication defective can be transly by helping virus to obtain complementary function, and this discovery can not realize the separation of the carrier of replication defective by oneself.On the contrary, because the existence of the essential help carrier that copy function is provided can cause the pollution in the preparation.Therefore an additional element must be used for introducing the specificity that the replication defective carrier duplicates or packs.Obtain in the packaging function of this element provided by the present invention by adenovirus.

There is the parcel signal of an adenovirus in the left end of the adenoviral gene figure that has pointed out in routine.Studies show that of a nearly step, genome E1A (194-358bp) even district disappearance mutant in the clone that early stage (E1A) function of complementary can be provided, also grow very slowly.When the compensation DNA (0-353) of one section adenovirus of reorganization arrived the right end of mutant, virus can normally be packed.Further mutation analysis obtains a short repetition element relevant with the position at the left-end point of genome Ad5.If a single copy finding this tumor-necrosis factor glycoproteins is present in genomic any end and can both causes enough effectively packing, but can not be moved into the inside of Ad5 DNA.

Use the packaging signal of different sudden changes just might obtain having the help virus of Different Package efficient.Usually, these sport point mutation or deletion mutantion.When the help virus of low packaging efficiency is helping to grow in the cell, although with wild-type mutually specific efficiency reduce, still can obtain packing, make to help the breeding of virus to realize.When these helped virus to grow in cell with the virus that contains the wild-type packaging signal, it is preferentially selected that the packaging signal of wild-type suddenlys change relatively.Under the condition that the limited packing factor exists, the virus that contains the wild-type signal helps virus optionally to be packed relatively.If selectivity is enough high, just can reach the product of homogeneous.B) retrovirus

Retrovirus is the RNA viruses of a class strand, and can be in cells infected their RNA being transformed into double-stranded DNA by the reverse transcription process with it is feature.The DNA product stably is incorporated into becomes provirus on the cell chromosome, guide the synthetic of viral protein.Integrated results remains virogene in being subjected to chief cell and offspring thereof.Retrovirus contains three genoid gag, and pol and env-be coded housing albumen, polymerase protein and envelope protein respectively.A sequence in that the gag upstream region of gene finds is designated as ψ, plays the function that genome is packaged into the signal of virus particle.Virus genomic 5 ' and 3 ' end two long terminal repeats (LTR) are arranged, contain strong promotor and enhancer sequence, also be integrated in the host cell necessary.

For making up a retroviral vector, the nucleotide sequence of a coding promotor is inserted into virus genomic appropriate location, obtains a virus of duplicating inactivation.For obtaining virus particle, made up one and contained gag, pol and env gene but do not contain LTR and the parcel clone of ψ part.When the plasmid of the reorganization of the cDNA that has the people and retroviral LTR and psi sequence is introduced in (as using the calcium phosphate precipitation method) in this clone, psi sequence make recombinant plasmid transcribe RNA be packed into virus particle, be secreted in the substratum again.The substratum that contains recombinant retrovirus is collected, and suitably concentrates, and is used for gene transformation.Retroviral vector can infect the cell type of wide scope.But many retroviral integration and stably express require the division of host cell.

By add the chemical modification method of galactose residue based on chemical on retrovirus envelope, realized making the design of retroviral infection specific objective recently.This modification is the same with the desire phase, allows via the specific cell of the receptor-mediated infection of asialoglycoprotein, as liver cell.

Another realizes that specific guide is a antibody by a biotinylated antiretroviral envelope protein and anti-specific cells acceptor to the recombinant retrovirus design.Antibody combines by using streptavidin (Roux etc., Proc.Natl Acad.Sci.USA, 86:9079-9083,1989) with vitamin H.By mainly organizing complex and the antigenic antibody of II, infect a series of different people's cells that have these surface antigens in external the displaying with cotropic virus with anti-.C) adeno associated virus

AAV is long linearity, the single-stranded DNA viruses of an about 4700bp.Both sides are for oppositely repeating end.Two genes are arranged in its genome, can produce some different gene products.One is the cap gene, produces three different virion proteins (VP), is designated as VP-1, VP-2, VP-3.Another is the rep gene, four Nonstructural Proteins (NS) of encoding.The product of one or more rep genes is responsible for the trans-activation that AAV transcribes.

Three promotors among the AAV are the unit classification by its position on genome with the map distance.Be p5, p19 and p40 from left to right.Transcribe six transcription products, each promotor starts two transcribes, one of them is sheared processing.It is identical that the shearing site that is positioned at map distance 42-46 is transcribed each.Four Nonstructural Proteins are obtained by long transcribing, and three virion proteins are obtained by transcribing of minimum.

AAV is not associated with any pathology stage of people.Interest be, AAV will effectively be duplicated, and needs " help " function that other virus provides, as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus, and as its described adenovirus.Adenovirus has been a feature solves the most clearly help virus, and its many early functions show it is to be used to assist duplicating of AAV.Be sure of that the proteic low expression level of rep among the AAV can end the expression of AAV composition, helped the infection of virus can remove this inhibition.

The terminal repeat of AAV carrier can by with restriction enzyme cutting AAV or contain as p201 a modified AAV genome (Sarnulski etc., J.Virol_61 (10): 3096-3101 is 1987, with reference to annex hereinafter) plasmid and obtain.Perhaps obtain, comprise based on the AAV sequence of having delivered and synthesize terminal repeat with chemistry or enzyme process by other known technology method, and other method that is not limited thereto.These conventional synthesis techniques and the method for knowing are analyzed the same ITR sequence that can detect AAV as deletion mutantion, bring into play it such as stable and the necessary least part of site-specific nature integration function.And can detect the small modification that this sequence can allow in the scope that keeps its stable and site-specific integration function.

Based on the carrier of AAV at the external safe face gene delivery means efficiently that is proved to be.And this class carrier reach in vivo the gene that the treatment potentiality are arranged that just is being applied to wide scope in being of deriving in the body carry out clinical before and the detection of clinical phase.

The effective conversion of AAV mediation has entered clinical trial (Carter and Flotte, Ann.N.Y.Acad Sci.700:79-90,1995 with expression in the treatment of cystic fibrosis in lung; Flotte etc., Proc.Natl.Acad.Sci.USA, 90:10613-10617,1993).Similarly, with the AAV mediation dystrophin gene is transferred in the skeletal muscle with the treatment muscular dystrophy as expected, reach tyrosine hydroxylase is conveyed in the brain with the treatment Parkinson's disease, factor is conveyed in the liver with the treatment hemophilia B, and have very much vascellum esoderma growth factor gene being imported in the heart of potentiality with the treatment myocardial infarction, because experiment recently shows that the transgenosis of AAV mediation can very high efficiency expression in these organs, has seemed it is feasible.(Fisher etc., J.Virol, 70:520-532,1996; Flotte etc., Proc.Natl.Acad.Sci.USA, 90:10613-10617,1993; Kaplitt etc., Nat.Genet_8:148-153,1994; Kaplitt etc., Arm Thord.Surg_62:1669-1676,1996; Koeberl etc., Proc.Natl.Acad.Sci.USA, 94:1426-1431,1997; McCown etc., BrainRes_713:99-107,1996; Ping etc., Micro circulation, 3:225-228,1996; Xiao etc., J.Virol_70:8098-8108,1996) d) other virus vector

Other virus vector can be used as the expression assembly in the present invention and is applied to.Comprise by vaccinia virus derive carrier (Ridgeway, In:Vectors:Asurvey of molecularcloning vectors and their uses, Rodriguez RL, Denhardt DT.Ed_Stoneham:Butterworth, pp.467-492,1988; Baichwal and Sugden, In:Gene Transfer, Kucherlapati R, ed_NewYork, PlenumPress, pp.117-148,1986; Coupar etc., Gene, 68:1-10,1988), reach carrier by sparrow poxvirus, slow virus, simplexvirus gained.

6. target cell

Method involved in the present invention and plasmid can with a series of cells in the Mammals, organ, and tissue as target.

In certain embodiments, expression construct described herein is used to cancer therapy.Target cell or be tumour cell perhaps is the cell of inside tumor or near cell.Tumour then can be arranged in brain, lungs, liver, spleen, kidney, bladder, lymphatic node, small intestine, pancreas, rectum, stomach, breast, uterus, prostate gland, testis, vaginal orifice, uterine cervix, ovary, skin, incidence, oesophagus, marrow or blood.The those skilled in the art in field of the present invention can detect the gene that is suitable for treating usefulness to be expressed in given tumor type at an easy rate.

In further embodiments, be applied to treat the pharmacology situation of non-cancer.For example, the alternative medicine of albumen efficiently provided by the invention.In this example, the cell of particular type, tissue and organ can be used as at certain proteic target of patient's expression in vivo, and especially this proteic activity only is restricted in this specific cell, tissue or the organ.Same, which cell the those skilled in the art in field of the present invention can detect at an easy rate is suitable for as target most very much.

Can expression construct be incorporated in the interested cell of institute by external, ex vivo or intravital method.Because transfection or transduction isolated cells are in general much effective, the method for ex vivo has been adopted in present many gene therapies.The selection of introduction method and specific transport vehicle will be according to the type of target cell, tissue or organ.The those skilled in the art in field of the present invention can instruct chosen process simply.

Because the expression construct among the present invention need can be activated through inducing, expression construct can be transferred in a big way the position of patient body in many examples, rather than imagination only is specific cell, tissue or organ.Limit the patient is exposed under the activation condition of abduction delivering construct expression, to obtain the expression of specificity.In many examples, this is best.For example, the patient is exposed to when treating in the ray, is limited to the position of having only those to shine.The application of hot temperature therapy also must limit its scope.In some other embodiment, reactive conditions be exactly target cell itself specificly have.For instance, the low-oxygen environment that tumour had can activate the expression with the expression construct that contains derivable hypoxemia reactive element promotor.In such example, the expression that is caused will very realize compartmentation naturally, although the introducing of expression construct may not be compartmentation.

7. partner treatment

Expression construct involved in the present invention has the advantage that can combine with one or more other traditional methods of treatment.A target of cancer therapy now is exactly to seek the approach that can improve chemotherapy and radiotherapy efficient.One can combine traditional treatment with gene therapy.For example, herpes simplex thymidine kinase (HS-tk) gene, when its after the retroviral vector system is introduced in the brain tumor, can successfully induce the antagonism viral reagent guanine susceptibility.But the required clinical effective expression amount that reaches was limited to after gene therapy cooperated the efficient of other traditional remedies still to be transferred to target cell by gene.

Want cell killing, growth of containment cell and metabolism, reduce the size of tumour, or the phenotype of reverse or the pernicious amplification of reduction tumour cell, with method provided by the invention and assembly, just can prevailingly expression construct of the present invention be imported in the target cell and and obtain abduction delivering by application heat shock or other condition that can activate inducible promoters.This gene therapy can be used with other composition that contains the cancer therapy effective agents.These compositions should use corresponding dosage so that can kill or suppress the amplification of cell effectively.This process can adopt expression construct and corresponding medicament or factor while transfered cell.Can perhaps cell be exposed to simultaneously two different component or prescriptions by allowing single composition or the medicinal prescription that contains the two of cells contacting, one contains expression construct, and another contains other medicament of using.

Gene therapy in addition/heat shock treatment also can in advance or lag behind other pharmaceutical treatment, at interval can be from several minutes to several weeks.In certain embodiments, other medicament and expression construct are separated to be applied on the cell, one usually can keep do not lose efficacy at the compartment of each importing guaranteeing sufficiently long action time, so medicament and expression construct still can realize the advantage of pair cell partner treatment.In these examples, it is contemplated that its just like will acting on when in 12-24 hour or preferable 6-12 hour, containing the cell of two kinds of binding modes simultaneously, only taking, be first-selected 12 hours time of lag.In some cases, can expect can extend between each independent usage last several days (2,3,4,5,6 or 7) at interval during the treatment, or even several week (1,2,3,4,5,6,7 or 8).

It is contemplated that equally also can expect has more than a kind of using method between expression construct and medicament.Various possible fit systems all may be applied to, and as with " A " expression expression construct, represent another medicament with " B ", can be exemplified below:

A/B/A????B/A/B??B/B/A??A/A/B??B/A/A??A/B/B??B/B/B/A??B/B/A/B

A/A/B/B??A/B/A/B??A/B/B/A??B/B/A/A??B/A/B/A??B/A/A/B??B/B/B/A

A/A/A/B??B/A/A/A??A/B/A/A??A/A/B/A??A/B/B/B??B/A/B/B??B/B/A/B

Similarly, other fit system also is expected.Illustrate again, reach the purpose of cell killing, the two will use effective cooperation dosage transfered cell of energy cell killing.

The medicament or the factor that are suitable for partner treatment have: any chemical agent or methods of treatment that can the inducing DNA damage when being applied on the cell.The such medicament or the factor comprise the radioactive rays or the hertzian wave of energy inducing DNA damage, as gamma-radiation, X-ray, uv irradiating, microwave, electron rays etc.

In one embodiment of the invention, the radiotherapy that cooperates with gene therapy is made up of the bundle of rays of outside.External-beam is generally the ray of taking high energy, as high energy x-bundle of rays.

Another approach that can be used with gene therapy is an inner radiation, or shor time treatment.The method of carrying out shor time treatment comprises that an inner chamber or a matter places radioactive source, and perfusion hydrosol, parenteral or oral area are taken etc.The source of closure is wrapped in metal, lead, pipe, pin or other analogue.Nonocclusive radioactive source is prepared into aaerosol solution.

The radioactive element that parcel is good is placed in the body cavity, or directly inserts in the tissue with appropriate device.Device is generally put into body cavity or tissue with surgical method or is used the method for fluorescence.These devices are generally plastics or metallic sheath, can be sewn into and maintain the position near tumour.Radioactive isotropic substance just is placed into (load the back) in the device after a while.Radioactivity is implanted to be used for the treatment of and is positioned at tongue, lip, breast, vagina, uterine cervix, uterus, rectum, bladder, and the cancer of brain.The source that parcel is good also can be stayed in the patient body as nonvolatil implantation.The bead that implantation is made up of radioactive substance is a treatment position property prostate cancer, and position property but an approach of the lung cancer that is unsuitable for performing the operation.

The chemical substance of a series of tool inducing DNA damage functions also is described and makes chemotherapeutic, all can be used from partner treatment with the method one that this paper proposes.Imagine available compound include as, Zorubicin, 5 FU 5 fluorouracil (5FU), etoposide (VP-16), camptothecine, actinomycin D, ametycin, cisplatin (CDDP), and even available hydrogen peroxide.The present invention has similarly comprised and has been used one or more dna damage reagent simultaneously, no matter be based on compound ray or real, for example the X-ray used with cisplatin, or uses cisplatin and etoposide simultaneously.

For instance, when treating cancer, we can allow tumour cell contact medicament according to the present invention, and the expression construct of affix is simultaneously also passed through the heat-inducible expression of gene.This can be by carrying out local heat shock or the whole body heat shock of individuality being realized to tumor locus.This process can cooperate with the radio exposure to tumour carries out, and the available ray has such as X-ray, UV-light, gamma-radiation, so that microwave.Tumour cell contacts other medicament, and also by allowing patient take a certain amount of medicinal component that treatment is renderd a service, the compound that comprises has Zorubicin, 5 FU 5 fluorouracil, etoposide, camptothecine, actinomycin D, ametycin, and the more normal cisplatin of getting.These medicaments can be prepared to the mode of similar partner treatment component or test kit, and are cooperating with the expression construct that is used for the treatment of as described and use.

Those can cause nucleic acid especially the medicament of DNA commissure also be conceived to be used for auxiliary dna damage to obtain cooperating the common antitumous effect of expression construct of the present invention.Such medicament is just like cisplatin and other DNA alkylating agent.Cisplatin can not pass through oral absorption, therefore must by in vein, subcutaneous, tumour or peritoneal injection transmit.

The medicament of other energy damage dna also comprises the compound that can disturb dna replication dna, mitotic division, chromosome segregation.These chemotherapy materials comprise Zorubicin, etoposide, isoptin, podophyllotoxin, and other analogue.When clinical treatment tumour widely, the absorption of these compounds is by heavy dose of intravenous injection, dosage can from Zorubicin every 21 days 25-75mg/m ²100mg/m to etoposide ²Or this dosage by oral twice.

Disturb nucleotide precursor reagent synthetic or reliability also can cause the damage of DNA.So developed the precursor of many Nucleotide.Those have obtained extensive testing and the precursor that is easy to obtain is particularly useful.Such medicament uses 5-FU just like 5 FU 5 fluorouracil (5-FU) because tumour cell is partial to preferential the selection, makes it be particularly conducive to and is applied on the tumour cell.Although the strong toxicity of 5-FU tool, it is still used many-sidedly, even comprises that dosage range commonly used is 450-1000mg/m ²The vein in/sky is taken in.

The factor that can cause dna damage that other wide model is used includes reaching such as gamma-radiation, X-ray of knowing and directly imports radio isotope in tumour cell.Also the factor that can be susceptible to has microwave, UV-light etc.Probably, all these factors, effect produces the DNA of wide scope, DNA precursor, dna replication dna and reparation, and the damage in chromosomal assembling and the maintenance.The irradiation dose of X-ray can from every day 50-200 roentgen continue for some time (3 to 4 week) to the dosage that shines 2000-6000 roentgen once.Radioisotopic using dosage scope is wider, mainly according to the kind of isotopic transformation period, radioactive rays and intensity, and the absorbing state of tumour cell.

The technology that relates to can be with reference to " Remington ' s Pharmaceutical Sciences ", and the 15th edition, 33 chapters are mainly at the 624-652 page or leaf.Using dosage must make corresponding changes according to the condition of patient's object.Everyone reaction of taking allows that situation has determined each patient's suitable dose in all situations.Prior, when being applied to human body, prepared product will reach official's biological standard of FDA on aseptic, thermal source, security and purity.

To cancer patient local import the expression construct that is used for the treatment of of the present invention be import anti-at the method that can preferentially select of treatment effective gene of clinical disease.Similarly, chemistry and radiation therapy method can be directed to the specific affected position of patient.In addition, general ground imports expression construct and medicament is suitable in some cases, for example when tumorigenic whole body diffusion.

Outside gene therapy combined with chemotherapy and radiotherapy, also it is contemplated that the superiority of polygene partner treatment.For example, import the anticancer effect that p53 and p16 mutator gene can obtain reinforcement simultaneously.All other tumor-related genes all can be imagined with this mode and import, and include: p21, Rb, APC, DCC, NF-1, NF-2, BRCA2, p16, FHIT, WT-1, MEN-1, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl, and abl.

Medicinal compositions with take in the path

The absorption mode that the composition that the present invention is used for the treatment of can be imagined has via external, from intravital, and intravital.Therefore to may application mode preparation of compositions be become corresponding suitable medicinal compositions according to each.Usually, this requires the medicinal compositions of preparation must not have heat source substance, and other might hurt human or animal's impurity.Equally also can use suitable salt and damping fluid to keep the stable of composition and to make target cell take in this composition.

Composition of the present invention comprises the effectively expressing construct of some amount or the expression construct of being carried by virus vector or liposome, and it is dissolved or dispersed in the medicinal acceptable carrier or the aqueous solution.These compositions also can provide by the mode of inoculation." medicinal " or " acceptable on the pharmacology " is meant that molecule integral body and composition can not produce reaction deleterious on the contrary, hypersensitive or that other is not expected when correctly taking among the human or animal." acceptable carrier on the pharmacology " comprises any and whole solution herein, dispersion medium, outsourcing capsid, antibiotic and antifungal medicine, isoosmotic and absorption delay reagent, and other similar carrier.The those skilled in the art in field of the present invention are familiar with the application of these media and reagent very much.Except those and incompatible conventional media and the reagent of activeconstituents, be expected as therapeutic composition with them.Composition with auxiliary activity also can join in the composition.

Acceptable salt can join and tensio-active agent on alkali or the pharmacology, as hydroxypropylcellulose, suitably in the blended water with the solution of preparation active composition.The dispersion of composition also can be at glycerine, polyoxyethylene glycol liquid, and other similar mixtures, and carry out in the oil.When preserving with use under normal condition, these prepared products contain inhibitor to suppress microbial growth.

The mentioned therapeutic composition of the present invention also can comprise medicinal preparation and the human body absorption in the classical leading treatment.The absorption of the therapeutic composition that the present invention is mentioned can be via the approach of any routine, as long as can arrive destination organization or cell by this approach.This comprises via oral area, nose, cheek, rectum, vagina and partial.The injection of other absorption has coordination, intracutaneous, subcutaneous, intramuscular, endoperitoneal or vein.These compositions are ingested with medicinal acceptable manner usually, comprise physiology acceptable carrier, damping fluid or other vehicle.Be applied to when antitumor, can imagine directly to inside tumor injection or the tumour kitchen range that excise, regionality (for example lymph) or general.Also it is contemplated that implementation to focus,, poured into continuously last several hours to several days by conduit as tumour or tumour kitchen range.

Therapeutic composition of the present invention is beneficial in the injectable mode of the aqueous solution or suspension and takes in; Also can be prepared into the solid mode that is suitable for dissolving or being suspended in injection liquid.The preparation form also can be emulsion.Typically, the composition that is used for this purpose comprises medicinal acceptable carrier.For example, said composition can be the phosphate buffered saline buffer that contains every milliliter of about 100mg.Other medicinal acceptable carrier has the aqueous solution, non-toxic excipients, and salt, and sanitas, damping fluid and other analogue all might be used to.Nonaqueous example has propylene glycol, polyoxyethylene glycol, and vegetable oil reaches organic ester such as ethyl oleic acid ester that injectable is used.Aqueous carrier comprises water, ethanol/water solution, salts solution, carrier that non-enteron aisle is used such as sodium-chlor and woods Ge Shi Glucose Liquid etc.Vein delivery carrier includes liquid and nutritional supplement.Sanitas comprises microbiotic, antioxidant, sequestrant, and inert gas.The pH value of the composition of various medicinal compositionss is adjusted to the constant of knowing with concrete concentration.

Also imagined in addition and be suitable for the prescription that oral area is taken in.Oral prescription has comprised classical vehicle, the mannitol of pharmaceutical grade for example, lactose, starch, Magnesium Stearate, asccharin sodium salt, Mierocrystalline cellulose, magnesiumcarbonate, and other analogue.The existence form of composition is solution, outstanding solution, tablet, pill, capsule, the lasting pulvis prescription that discharges.When taking classical pathway, form can be emulsion, ointment, ointment or sprays.

The decision of the effective dose of healing potion will be based on desired target, for example: (ⅰ) suppress the tumour cell amplification, (ⅱ) remove or the kill tumor cell, perhaps (ⅲ) transforms the therapeutic gene of a short-term or long-term expression.Term " unitary dose " refers to the separation unit physically that is suitable for using on the patient.Per unit contains the quantity of a pre-set treatment component, is used to calculate the reaction that produces expection, and these reactions are as discussed above to be associated with dominating to treat with absorption method such as suitable pathways.The amount of required usefulness will be according to the number of times and the unitary dose of treatment, also will be according to by the patient, patient's state and the result of expectation that are treated.The present invention has also imagined the influence of polygene treatment.

In one embodiment, the carrier of coding one therapeutic gene is used to treat cancer patient.The typical amounts that is used for the treatment of the virus vector of cancer is 10 ⁶-10 ¹⁵The PFU/ agent (as, 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹, 10 ¹⁰, 10 ¹¹, 10 ¹², 10 ¹³, 10 ¹⁴And 10 ¹⁵), this dosage is divided into several parts of different sites injections in the solidity tumour.Therapeutic modality also relates to some transforming genes of taking in periodically, and interim is 3-10 week.For obtaining the benefit of continuity treatment, taking in carrier for more time also needs, and the time length can be some months by several years.

In another embodiment of the present invention, the virus vector of coding one therapeutic gene can be used to people or Mammals inoculation.Typically, in this inoculation embodiment, people or Mammals will be taken in the some amount virus particle that is used to produce desired result, to keep a long-term expression of transforming gene, make the host can produce immune response.Can expect needs series of injections, and for example, with an elementary immunization, then twice booster immunization injection is so that can enough induce a secular immune response.Typical dosage can be 106 to 1015PFU/ injections according to desired result.The antigen of low dosage is induced strong cell-mediated reaction usually, and high dosage antigen is induced antibody-mediated immune response usually.The accurate amount of treatment component is also judged according to each specific practitioner.

9. example

Below each object lesson just be used to set forth the present invention, rather than to the restriction of claim scope.

Example one

Heat-inducible promoter is induced the expression of reporter gene

Carrier structure: be the ability of research HSP70 promotor inducible gene expression, with a minimized HS promotor (heat shock, heatshock, HS) or a minimized CMV promotor be inserted into the upstream of a reporter gene, reporter gene is positioned on the plasmid of a band Xin Meisu and penicillin selective marker.The design of the base of plasmid M5 as shown in Figure 1, it contains a multiple clone site on the operation site by the promotor of HSP70 gained.The structure of M5 is with pcDNA3.0 plasmid (Invitrogen, Inc.) the CMV promotor on is with a minimized HSP70B promotor (SEQ ID NO:1, Figure 10) substitute, it is a long 0.4kb fragment (Hind III-BamH I) that obtains by people's heat shock protein 70B (HSP70B) promotor, from StressGen, the Inc. place obtains.

The activity of HS and CMV promotor is by its cancer cells with S8 plasmid transfection people, and human breast cancer cell MCF7 and prostate cancer cell DU145 judge.The S8 plasmid is derived by M5 carrier (see figure 1), contains to be connected to the green fluorescent protein that coding strengthens (Enhanced Green Fluorescence Protein, HSP70B EGFP) minimizes promotor.The structure of S8 be with the pEGFP-1 plasmid (Clonetech, Inc.) the EGFP gene on be inserted into multiple clone site on the M5 (multiple cloningsite, MCS).

Cell culture medium and transfection: with aforesaid S8 plasmid transfection people's DU-145 prostate cancer derived cell and MCF-7 mammary cancer derived cell.For being separated to the S8 transfectional cell, the substratum calcium phosphate precipitation method transfection of standard.The cell that contains integrated plasmid comes out so that its ability that can grow in the presence of Xin Meisu is screened.Heat shock realizes in (± 0.1 ℃) by culturing bottle is immersed in the water-bath controller fully.

With Geneticin in the MCF7 cell transfecting, screen a positive colony, clone 4 and one polyclone system.In the DU-145 cell transfecting, screen to such an extent that a polyclone is.(in each example, cell screened for 2 weeks with Geneticin) screen clone with facs analysis and screening.

The separation of positive cell line: under heat shock response, express the cell of high-level EGFP by obtaining with traditional serial dilution method and fluorescence-activated cell sorting method (FACS).Count the expression of quantitative EGFP with wandering cells.Strong green fluorescent protein (EGFP) is activated at the 490nm place, makes it can be under fluorescent microscope observed or use facs analysis.Express the cell and the cell of not expressing EGFP FACS method sorting of EGFP.This step be to Geneticin screen clone carry out.The reason that must do like this is that in a polyclone clone, some is that the S8 plasmid can be integrated in a kind of mode of disturbing reporter gene to express.Get rid of this part cell, only stay pure positive part and be used for further analysis through sorting.

As shown in Figure 2, when growing in 37 ℃ with the DU-145 cell that contains EGFP plasmid (S8) stable conversion that minimizes the heat-inducible promoter driving, the mean fluorecence amount is approximate 10 relative fluorescence units.When cellular exposure behind 4 hours of 42 ℃ of heat shocks 1 hour, average relative fluorescence has 7-9 raising doubly.Corresponding genetic expression can change institute quantitatively by relative fluorescence in the measurement stable transfected cells simultaneously.With the MCF7 cell of FACS method sorting S8 plasmid transfection as shown in Figure 3.

Dynamics research: the suitableeest time/temperature that is used to obtain heating the MCF7 cell and does not cause the cell mass mortality is studied in the high temperature exposure survival.Under 40 ℃ and 42 ℃, necrocytosis only is insignificant less than 3% when growing by 1 hour.In the time of 44 ℃, only need almost 50% just death of cell in 30 fens.

Use the suitableeest survival time as above, carried out preliminary dynamics research.Detect with the FACS method, in the MCF7 of 40 ℃ and 42 ℃ following heat shock transfections cell 1 hour, heat shock 1 hour was than only producing more EGFP in 30 minutes.Be 4 hours the suitableeest time of recovery after the cell heat shock.Increase the raising that can not cause the EGFP expression level arbitrarily time of recovery.After 30 minutes, be longer 8 hours to reach the highest recovery 44 ℃ of heat shocks.

Different clones are in the expression of 37 ℃ of-44 ℃ of following EGFP:

Below identical in used heat shock/time of recovery and the dynamics research, in order to detect the derive inducibility of EGFP of promoters driven of in all clones that get by the transfection of invention group HSP70-.

40 ℃ of heat shocks in-1 hour recovered in 4 hours

42 ℃ of heat shocks in-1 hour recovered in 4 hours

44 ℃ of heat shocks in-30 fens recovered in 8 hours

Use these temperature and times, detected the expression of the EGFP of following clone.

MCF7: the clone that is derived from breast cancer cell.

DU145: the clone that is derived from prostate cancer cell.

The MCF7 cell of MCF7-S8-P:S8 plasmid transfection, polyclone clone.

MCF7-S8-PS1: through FACS sorting EGFP expression MCF7-S8-P clone once.

MCF7-S8-PS2: through the FACS MCF7-S8-P1 clone expressed of sorting EGFP again.

The clone 4 of MCF7-S8-4:MCF7 S8 transfection.

MCF7-S8-4S1: sorting once is used for the MCF7-S8-4 clone that EGFP expresses.

The DU145 cell of DU145-S8-P:S8 plasmid transfection, polyclone clone.

With HSP70 derive promoters driven EGFP cells transfected system expression data as shown in Figure 4.Corresponding EGFP content also is improved when temperature improves.These data show that the heat-inducible promoter of invention group responds to heat shock really.Though, in the time of 37 ℃, also still have the expression of EGFP.

The expression of EGFP after the DU-145 cell heat shock of stable transfection: inducing of endogenous heat-inducible promoter is temporary and is temperature correlation.When with minimize EGFP stable transfection that heat-inducible promoter drives and after the DU-145 of twice screening of FACS cell (DU-145-PS2 cell) is with a series of different times and temperature heat shock, the expression of reporter gene is that temperature relies on, and the maximum of just temporarily keeping after inducing pressure 15-24 hour is expressed (Fig. 5).These results show, keep momently under the condition that this promotor is just used herein activating, and EGFP are unsettled that fluorescence in the cell began to reduce after 15-24 hour.Activity at 40 ℃ of heat shocks minimized heat-inducible promoter after 1 hour or 2 hours has temporarily increased approximate 3 times.42 ℃ of heat shocks 1 or after 2 hours promoter activity increased by 13 and 25 times respectively.

EGFP expression ratio under the control of HS promotor and CMV promotor is: the data of Fig. 6 show that the activity of minimized heat-inducible promoter in the DU-S8-PS2 cell changes according to temperature variation in 37-43 ℃ of scope.Compare down, use the V9 plasmid,, have than with minimizing the heat-inducible promoter transfection and exceeding 50% promoter activity at the same cell of 43 ℃ of following heat-inducibles with the carrier (Fig. 7) that CMV promoters driven EGFP expresses, the DU-145 cell of stable transfection.The activity of CMV promotor obviously is not subjected to Temperature Influence in these cells.The temperature dependency of minimized HS promotor is not peculiar to the DU-145 cell.

Example two

Expression with HIV promotor in the construct and tat amplification IL-2

Initialize amplification research: carried out relating to the research of the construct that the new therapeutic gene that can increase expresses herein.For illustrating the principle of amplicon, several constructs have been made up.Construct also comprises a composition promotor outside the heat shock inducible promoters, a CMV promotor.These constructs are designated as plasmid L-27, X14, RR13, Y15 and SS10.Following table 3 shown each plasmid with promotor/gene and the expression amount of IL-2.Four plasmids wherein are to be obtained by a plasmid derivative that contains two multiple clone site.In these four plasmids, the CMV promotor is inserted into the upstream of a tat gene or a multiple clone site (MCS), and the upstream that the long terminal repeat (LTRs) of HIV1 and HIV2 one is inserted into mouse IF2 gene (IL-2).The structure of plasmid X14 and Y15 is shown in Fig. 9 A and 9B.The L-27 plasmid is as object of reference.(Medgenix Diagnostics, Fleurus Belgium) organize the method for culture supernatant to measure IL-2 content by detecting with ELISA with the IL-2EASIA test kit.The IL-2/ml into 0.1IU is estimated in the sensitivity of test kit.In this research, come transfection SW480 cell (, seeing below) referring to the transfection scheme in the example three with Dosper fat.

Table 3

Plasmid	Promotor/gene	The IL-2 amount, the I.U. of unit
When only having fat to exist	Dosper	4.8
L-27	CMV/IL-2	15.63
RR13	HIV1/IL-2 CMV/ multiple clone site	17.56
X14	HIV1/IL-2 CMV/TAT	173.7
SS10	HIV2/IL-2 CMV/ multiple clone site	12.83
Y15	HIV2/IL-2 CMV/TAT	440.55

Can improve expression of gene after the CMV promotor from the complete as can be seen amplification construct of this research.The proteic expression of trans-activating factor TAT simultaneously also is that raising is expressed necessary.

But the amplicon of example three heat-inducibles

Vector construction: use the activity that whether can improve minimized heat-inducible promoter for judging second promotor, with a series of different carriers transfection MCF-7 cell temporarily, carrier comprises pC8, and pf12 is with p007 (Fig. 8 and Fig. 9).By using a plasmid that contains two multiple clone site, minimized heat-inducible promoter is inserted into the upstream in a tat gene or a polyclone cell site, and the long terminal repeat (LTRs) of HIV1 or HIV2 is inserted into the upstream of mouse IF2 (IL-2) gene.Each plasmid has Xin Meisu and penbritin selective marker simultaneously.

At first will on plasmid C5, cut out contain IF2 (IL-2) coding region (see the GenBank database file, the EcoR I fragment of long 0.5kb no.577834) be inserted in the EcoR I site of M5 carrier (referring to the described example of example above) plasmid f11.The structure of plasmid C8 be with from plasmid B4527 (referring to Tsang etc., Biotechniques 20:51-52,1996 with Tsang etc., Biotechniques 22:68,1997, the two is listed in the reference in this article) MCS site upstream cut out one to contain the long segmental length of HSP70B of 0.4kb be that the BamH I fragment of 1kb is inserted into plasmid DNP-1 (Tsang etc., 1996, with Tsang etc., 1997) on BamH I site in, the IL-2 upstream of coding region on the DNP-1 contains the LTR sequence of HIV1.Then will contain the Not I site that the long Not I of the 0.4kb fragment of HIV tat gene broadcasts on the C8 and get carrier fl2 (Fig. 9).The structure of the carrier D10 of intermediary is to contain the long BamH I fragment of 1kb that minimizes the HSP70B promotor with one to be inserted into plasmid MNP-7 (Tsang etc., 1996 with Tsang etc., 1997), and the upstream of the last IL-2 of MNP-7 coding region has the LTR sequence of HIV2.The structure of plasmid 007 (Fig. 9) is that a long Not I fragment of 0.4kb of encoding the tat gene is inserted in the Not I site of D10.

The transfection scheme: transfection process is according to a scheme of having delivered (Stopeck etc., CancerGene Therapy, 5:119-126,1998).The MCF-7 cell is put in one 6 holes or 12 well culture plates and cultivates.Second day, cell added 1ml transfection liquid after using the rinsing of Hanks salt buffer solution.Transfection liquid is 4: 1 fat and the serum-free OptiMEM of DNA (from Gibco BRL) for containing mass ratio, fat is Dosper (1,3-pair-oily carboxyl-2-(6-carboxyl-essence group base)-propyl amides, from Boehringer Mannheim) or Dmrie C (1, the two methyl of the two tetradecyloxyaniline propyl group-3-of 2--hydroxyl bromination ethyl ammonium, from GibcoBRL), plasmid DNA is 1.25 μ g or 2.5 μ g.In every hole, add Bovine Placenta serum (FBS) immediately to final concentration 10% (volume/volume).DmrieC is better than Dosper after testing.Cell before detecting IL-2 content, to before heat shock, cultivate 24 hours and heat shock after 24 hours.Thermoinducible amplification research: in a cover experiment, analyzed pC8, pf12, or the IL-2 expression activity of the cell of p007 plasmid transfection.(Medgenix Diagnostics Belgium) organizes culture supernatant to obtain with the detection of ELISA method to the amount of IL-2 by using IL-2 EASIA test kit.The sensitivity of test kit is estimated and is 0.1IUIL-2/ml.From then on the data that cover experiment obtains are shown in following table four.This table has shown the expression level of IL-2 in the MCF7 cell, and the MCF7 cell is used Dosper transfection post-heating 24 hours, uses elisa assay again behind 49 hours of transfection.Plasmid L-27 (be used for as the IL-2 of CMV promoters driven express with reference to plasmid), 007, f12 and C8 are detected.

Table 4

	The IL-2 activity, the I.U. of unit
Temperature						37℃	39℃	41℃	42℃	44℃
The heat shock time length	Continue	Continue	1 hour	1 hour	0.5 hour
Only there is L-27 007 F12 C8 in fat	2.03 14.2 336.76 8.40 9.19	0.50 9.88 318.49 6.88 8.03	0.41 5.95 334.02 49.93 11.74	0.53 9.88 373.74 60.02 8.73	0.53 7.80 389.27 88.13 16.37

From then in the research as can be seen pf12 heat shock is responded, and the IL-2 that makes up of the heat shock amplicon that produces than pC8 or pL-27 produce much more.Under 37 ℃, pf12 than CMV drive with reference to plasmid, L-27, fecund is given birth to 5 times IL-2.When cell spends the night 39 ℃ of following heat shocks, the CMV of pf12 than 37 ℃ drives the contrast multilist and reaches 7 times IL-2.Handled 1 hour in the following heat shocks of 41 ℃ or 42 ℃, can make the expression of amplicon construct compare CMV and drive the expression of carrier 37 ℃ under and have and reach 26 times raising.(the p007 plasmid almost reaches the activity of its maximum in the time of 37 ℃, so heat shock can not improve expression amount significantly).Pf12 also is in a high-caliber active condition in the time of 37 ℃.These results show that the scheme of amplicon can improve the level of genetic expression between about 37 ℃ and about 42 ℃.

In another set of different experiment,, illustrated the variation in the effective body of transfection by carrying the method for control plasmid co-transfection that the upstream is the beta-galactosidase gene of CMV promotor with one.The general approach of these experiments is that commentaries on classics subtituted culturing cell transfectional cell after 24 hours is cultivated heat shock substratum after 24 hours again, changes substratum and also cultivates the expression level of measuring IL-2 after 24 hours.As shown in table 5 below, the activity that CMV starts only is subjected to the very little influence of heat shock.Minimized heat-inducible promoter only has low-down activity when cell remains on 37 ℃, can be induced to 20 times raising after 42 ℃ of heat shocks.Seen at the cell of stable transfection, the activity of minimized heat-inducible promoter only has half of CMV promotor.

Table 5

IF2 (IL-2) expression amount ^*37 ℃ 42 ℃ of carrier promotors ^*Improve multiple (42/37) relative ratios ^{* *}L27 CMV-IL2 82.6 93.4 1.1 1.0 C8 HSP-MCS 84.7 70.6 0.8 1.9

HIV1-IL2f11????HSP-IL2?????2.3????54.0?????????23.7?????????????0.4(1)f12????HSP-TAT???107.6???347.4??????????3.2?????????????6.9(17)

HIV1-IL2007????HSP-TAT????747.5?1642.9??????????2.2????????????83.3(208)

???HIV2-IL2???????????????????????????????????????????????????

^*: IL-2 amount in every mg cell protein that numeric representation produced in 24 hours is a unit with IU

^*: heat shock 1 hour

^{* *}: based on 42 ℃ numerical value, CMV-β-gal cotransfection

When the HIV1 promotor is expressed at no tat, be similar to the CMV promotor and influenced by heat shock.But when minimized heat-inducible promoter was used to express tat, being expressed in after 42 ℃ of heat shocks of reporter gene improved sharp.When temporarily using heat-inducible promoter/tat and HIV1/IL-2 transfection, the expression of IL-2 is with similar when cultivating for 37 ℃ with heat-inducible promoter/MCS and HIV1/IL-2 cells transfected.This activity self has improved relatively and has surpassed 3 times and almost reach more than 7 times of CMV promotor after 42 ℃ of heat shocks.

When HS promotor/tat and HIV/IL-2 cells transfected maintain 37 ℃ or all shown the expression of substantial reporter gene after 42 ℃ of heat shocks.Do to exceed more than 80 times when metric relative promoter activity starts Individual existence than CMV with IL-2.Temperature adjusting decreases, and the expression of 42 ℃ of heat back reporter genes is approximate 2 times that same cell is kept 37 ℃ of culture expression amounts.

The reporter gene that temperature relies on is expressed the influence that not existed by second promotor.Shown in following table six, to express with the reporter gene that contains in the temporary cells transfected of the plasmid that minimizes heat-inducible promoter/tat and HIV2/IL-2, the mode with a kind of temperature dependent form between 37 ℃ to 44 ℃ improves.These results numerically at Fig. 4 only use the numerical value of the cell that minimizes the heat-inducible promoter stable transfection similar to seen in fig. 6.

Table 6

IL-2 expression amount (IU/ml) ^* 37 ℃ 39 ℃ 40 ℃ 41 ℃ 42 ℃ 44 ℃ of carrier promotorsC8 HSP-MCS 7.2 9.3 6.0 4.8 5.3 7.0

HIV1-IL2f12????HSP-TAT????40.6???--????--???--???133.1?--

HIV1-IL2007????HSP-TAT????224???222???230??250???375??470

???HIV2-IL2????????????????????????????????????

With shown in the temporary transfection MCF7 of carrier breast cancer cell; Heat shock is 1 hour after 24 hours; Collect substratum in 24 hours after the heat shock and detect IL2

The research of example four animal bodies

On feature on the histology and metastatic potential, be similar to the mouse model of the human cancer of people's tumour, can treat with therapeutic composition provided by the present invention.In one embodiment of the invention, use the human tumor cells injection SCID mouse of report construct stable transfection, the report construct contains the TAT of HSP70B promoters driven expression and the EGFP or the IL-2 of HIV-1 or the expression of HIV-2 promoters driven.When tumor growth during to suitable detectable size, as diameter 1cm, with ultrasonic heating tumour to 42 ℃ of temperature.The expression of the different time quantitate gene after heat shock, method are that tumor resection prepares tissue slice, detect the fluorescence from EGFP, perhaps with the IL-2 level in the ELISA method detection tumor tissues.Use another embodiment of the present invention, human tumor cells is injected in the SCID mouse.Tumor growth is injection DNA-fat complexes during to suitable detectable size.With ultrasonic heat shock tumour, detect genetic expression after the heat shock.The validity of treatment is reduced by tumor size, and the reduction of transfer activity, the reduction of cell amplification and the termination of tumor growth show, as the effect of taking in therapeutic composition of the present invention.

Under the situation of the scope of the invention and spirit, the those skilled in the art in field of the present invention can obtain various modification body of the present invention and variant.Although the present invention when describing and some specific reference examples interrelate, should be appreciated that claim of the present invention should not be restricted in these certain embodiments.In fact, the variety of way of described this invention of realization is obviously included within the scope of the appended claims.

Sequence table＜110〉Tsang, Tom

Gerner,Eugene?W.

Harris,David?T.

Hersh; Evan＜120〉＜130〉15907-0016＜140〉＜141〉＜150〉US 60/064,088＜151〉1997-11-03＜160〉1＜170〉Patent In Ver.2.0＜210〉1＜211〉469＜212〉DNA＜213〉Homo sapiens＜400〉1ggatcctcca cagccccggg gagaccttgc ctctaaagtt gctgcttttg cagctctgcc 60acaaccgcgc gtcctcagag ccagccggca ggagctagaa ccttccccgc gtttctttca 120gcagccctga gtcagaggcg ggctggcctt gcaagtagcc ccccagcctt cttcggtctc 180acggaccgat ccgcccgaac cttctcccgg ggtcagcgcc gcgctgcgcc gcccggctga 240ctcagcccgg gcgggcgggc gggaggctct cgactgggcg ggaaggtgcg ggaaggttcg 300cggcggcggg gtcggggaga gaaaccgcag ggagagcctc actgctgagc gcccctcgac 420gcgggcggca gcagcctccg tggcctccag catccgacaa gaagcttac 469

Claims (46)

1. the method for the selected polynucleotide of effective expression in mammalian cell is characterized in that described method comprises:

(a) provide a kind of expression construct, this expression construct comprises that (ⅰ) suitably is connected to the inducible promoters of the gene of coding trans-activating factor; And second promotor that (ⅱ) suitably is connected to described selected polynucleotide, wherein, this second promotor is activated by described trans-activating factor;

(b) import described expression construct in described cell; And

(c) this cell is positioned over can activates in the condition of described inducible promoters, wherein, described condition causes the expression of described selected polynucleotide.

2. method according to claim 1, its feature are that also described inducible promoters is a heat-inducible promoter, and the condition that activates this startup is a hot conditions.

3. method according to claim 2, its feature also are about basal temperature that described hot conditions comprises described cell temperature between about 42 ℃.

4. method according to claim 3, its feature are that also described hot conditions comprises the temperature between about 37 ℃ and about 42 ℃.

5. method according to claim 4, its feature are that also described hot conditions comprises the temperature between about 38 ℃ and about 41 ℃.

6. the method for advancing according to claim 5, its feature also be connect hot conditions and comprise temperature between about 39 ℃ and about 40 ℃.

7. method according to claim 2, its feature also are described heat-inducible promoter by HSP70, HSP90, and HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin, deriving with one of HSP28 promotor obtains.

8. method according to claim 1, its feature are that also described inducible promoters comprises the hypoxemia reactive element.

9. method according to claim 1, its feature are that also described second promotor is selected from one of HIV-1 promotor and HIV-2 promotor, and described trans-acting factor is tat.

10. method according to claim 1, its feature are that also the expression of wherein said polynucleotide produces a polypeptide, protein, ribozyme, or one section antisense nucleotide.

11. method according to claim 1, its feature also are described selected polynucleotide to encoding histone, this albumen is selected from and contains ornithine decarboxylase antizyme, p53, p16, neu, IL1, IL2, IL4, IL7, IL12, IL15, the FLT-3 part, GM-CSF, G-CSF, IFN γ, IFN α, TNF, HSV-TK, I-CAM1, HLA-B7 is with one of TIMP-3.

12. method according to claim 1, its feature are that also described expression construct also comprises the gene of the selective marker of encoding.

13. method according to claim 1, its feature are that also described expression construct comprises that also (ⅰ) suitably is connected to second selected polynucleotide on described second promotor; And (ⅱ) be positioned at internal ribosome recognition site between the described first selected polynucleotide and the second selected polynucleotide.

14. method according to claim 1, its feature are that also described cell is a tumour cell.

15. method according to claim 1, its feature also be, wherein, described expression construct imported in the described cell mediated by one of following transport vehicle: liposome, retrovirus, adenovirus, adeno associated virus, the sparrow poxvirus, hsv, and vaccinia virus.

16. method according to claim 1, its feature are that also wherein, it is to carry out external that described expression construct is imported in the described cell.

17. method according to claim 1, its feature are that also wherein, it is to carry out in vivo that described expression construct is imported in the described cell.

18. the method for the expression product of the tool treatment effective dose that selected polynucleotide is provided comprises:

(a) provide first expression construct, this construct comprises inducible promoter, and it is connected on the gene of a coding trans-activating factor suitably;

(b) provide second expression construct, this second expression construct comprises second promotor, and it is connected to described selected polynucleotide suitably, and wherein, this second promotor is activated by described trans-activating factor;

(c) described first and second expression construct is imported to by in the treatment target; And

(d) described cell is placed the condition that can activate described inducible promoters, wherein, this condition can be induced the expression of described selected polynucleotide.

19. method according to claim 18, its feature are that also described inducible promoters is a heat-inducible promoter, and activate this condition that can induce startup and comprise from about basal temperature to about 42 ℃.

20. method according to claim 19, its feature are that also described first and second expression construct is positioned on the identical carrier.

21. method according to claim 20, its feature also are, wherein, adopt the method for ex vivo that described expression construct is imported in the described cell.

22. method according to claim 18, its feature are that also wherein described expression construct being imported in the described cell is to carry out in vivo.

23. method according to claim 18, its feature are that also the expression product of wherein said selected polynucleotide has injury effect to a certain pathogenic agent, wherein, this pathogenic agent comprises virus, bacterium, and fungi is with parasite.

24. method according to claim 18, its feature are that also the expression product of wherein said selected polynucleotide can suppress the growth of described cell.

25. method according to claim 18, its feature are that also the expression product of wherein said selected polynucleotide can replace defective albumen.

26. method according to claim 18, its feature are that also the expression product of wherein said selected polynucleotide promotes the regeneration of nerve fiber.

27. the method for treatment mammalian cancer is characterized in that comprising the steps:

(a) provide expression construct, this expression construct comprises that (ⅰ) is connected on the inducible promoters of gene of coding trans-activating factor suitably; Reach second promotor that (ⅱ) is connected to suitably on the selected polynucleotide, wherein, this second promotor is activated by described trans-activating factor;

(b) this expression construct is imported in the tumour cell; And

(c) this tumour cell is placed the condition that can activate described inducible promoters, this condition causes the amount of the expression of described selected polynucleotide and selected polynucleotide expression product enough to suppress the growth of this tumour cell effectively herein.

28. method according to claim 27, its feature are that also wherein said inducible promoters is that heat-inducible promoter and the condition that activates this inducible promoters are hot conditions, comprise the temperature of about basal temperature between about 42 ℃.

29. method according to claim 27, its feature also are further to comprise: treat described tumour cell with at least a treatment method for cancer of having set up, described method is selected from external-beam treatment, built-in radioactive source, chemotherapy, and surgical method it

30. method according to claim 27, its feature also are further to comprise:

(d) after described tumour cell being placed under the hot conditions, treat this cell with ray protector WR-33278 or WR-1065; And

(e) in the end one go on foot, treat described tumour cell with the radiotherapy method, wherein said selected polynucleotide is encoded to the ornithine decarboxylase antizyme.

31. method according to claim 27, its feature are that also wherein said Mammals comprises the mankind.

32. method according to claim 27, its feature are that also described cancer is selected from the cancer at following position: brain, lung, liver, bladder, spleen, kidney, lymphoglandula, small intestine, pancreas, hemocyte, rectum, stomach, mammary gland, uterus, prostate gland, testis, ovary, skin, vaginal orifice, uterine cervix, incidence, esophagus, marrow, and blood.

33. one kind in a certain immunoreactive method of Mammals moderate stimulation, its feature also is to comprise:

(a) provide expression construct, this expression construct comprises that (ⅰ) is connected to the inducible promoters on the coding trans-activating factor gene suitably; Be connected to second promotor on the selected polynucleotide suitably with (ⅱ), wherein, this second promotor is activated by described trans-activating factor;

(b) this expression construct is imported in the mammalian cell; And

(c) this cell is positioned over activates in the condition of described inducible promoters, described condition causes the expression of described selected polynucleotide, and the expression amount of product enough causes immune response effectively in this Mammals, and this immune response is selected from one of humoral immune reaction and cell immune response.

34. method according to claim 33, its feature are that also described inducible promoters is that heat-inducible promoter and the condition that activates this promotor are hot conditions, comprise the temperature of about basal temperature between about 42 ℃.

35. method according to claim 33, its feature are that also described immune response purpose is anti-described cell.

36. method according to claim 35, its feature also are further to comprise: treat described cell with at least a treatment method for cancer of having set up, method is by being selected from: chemotherapy, external-beam treatment, built-in radioactive source, and surgical method.

37. method according to claim 33, its feature are that also described Mammals is for human.

38. a method that changes the mammalian genes material, its feature also are to comprise:

(a) provide expression construct, this expression construct contains (ⅰ) and is connected to inducible promoters on the coding trans-activating factor gene suitably; Be connected to second promotor on the selected polynucleotide suitably with (ⅱ), wherein, this second promotor is activated by described trans-activating factor;

(b) this expression construct is imported in the mammalian cell.

39. an expression construct comprises:

(a) gene of coding trans-activating factor;

(b) be connected to inducible promoters on this gene suitably;

(c) polynucleotide of selecting; And

(d) be connected to second promotor on the described selected polynucleotide suitably, this second promotor is activated by described trans-activating factor.

40. according to the described expression construct of claim 39, its feature is that also described inducible promoters is induced by hot conditions by the expression of heat-inducible promoter and described selected polynucleotide, and this hot conditions comprises the temperature of about basal temperature between about 42 ℃.

41. according to the described expression construct of claim 40, its feature also is described heat-inducible promoter by HSP70, HSP90, and HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin is with acquisition that one of HSP28 promotor is derived.

42. according to the described expression construct of claim 39, its feature is that also described inducible promoters comprises the hypoxemia reactive element.

43. according to the described expression construct of claim 39, its feature is that also described second promotor is selected from HIV-1 promotor and HIV-2 promotor, and described trans-acting factor is selected from tat.

44. according to the described expression construct of claim 39, the expression of wherein said polynucleotide produces polypeptide, protein, ribozyme, or one section antisense nucleotide.

45. according to the described expression construct of claim 39, its feature is that also described expression construct comprises that further (ⅰ) is connected to second selected polynucleotide on described second promotor suitably; And (ⅱ) be positioned at internal ribosome recognition site between described first and second selected polynucleotide.

46. cell that comprises expression construct as claimed in claim 39.

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