CN1326506A - Use of specific hybrid promoters for controlling tissue expression - Google Patents
Use of specific hybrid promoters for controlling tissue expression Download PDFInfo
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- CN1326506A CN1326506A CN99811334A CN99811334A CN1326506A CN 1326506 A CN1326506 A CN 1326506A CN 99811334 A CN99811334 A CN 99811334A CN 99811334 A CN99811334 A CN 99811334A CN 1326506 A CN1326506 A CN 1326506A
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Abstract
The invention concerns a hybrid promoter comprising: all or part of the enhancer region of a strong and ubiquitous promoter/enhancer; a promoter region enabling specific expression in smooth muscle cells, and all vector or cell containing said promoter, and their uses.
Description
The present invention relates to biological field, particularly the field of genetic expression adjusting.The present invention discloses novel constructs and the novel vector that is used for the directed strongly expressed of gene especially.The present invention can be applicable to many fields, especially for producing recombinant protein, being used to set up transgenic animal model, being used to set up clone, being used to carry out shaker test or being used for cell therapy and gene therapy.
Can regulate and control and instruct genetic expression to constitute the very important stake in the biotech development.External, this allows to utilize the specific cells group to improve the condition that recombinant protein is produced.External, this technology also makes it possible to detect or prove the existence of specific cells group in the sample, or checks the characteristic of certain product or the gene among the adjusting specific cells group.The regulation and control of genetic expression in the body or stripped gene therapy also be very important, can selective control the production of treatment molecule in this therapy, be vital.In fact, according to different application, according to gene to be transferred, the specific tissue or the specific part that can only be oriented to organism are very important so that concentrate curative effect, restriction diffusion and side effect.
Can realize the orientation expression of given nucleic acid by diverse ways.First method is for example to utilize carrier or the transfering reagent with given cell specificity.But the specificity of being brought by this carrier is generally quite extensive, can not be oriented to accurate cell mass.Another kind method is the expression signal that utilizes certain cell type special.To this, reported so-called " specificity " promotor in the document, the promotor of pyruvate kinase, villin, GFAP encoding gene for example, lipid acid is in conjunction with the promotor of Erepsin, the promotor of smooth muscle cell alpha Actinin, the SM22 promotor, the promotor of somebody's albumin gene.But, if the specific in a organized way words of these promotors, their effectiveness also relatively a little less than.For example, these promotors is most of active in so-called " by force " promotor, generally low 10-100 at least times.In addition, it is generally acknowledged that the specificity of promotor and its intensity are inverse ratio, intensity is big more, and non-specific activity level is also big more.
Therefore, it is particularly advantageous providing organizing specific and strong promotor.Purpose of the present invention should provide the novel constructs that is used for the directed strongly expressed of gene.
The present invention discloses especially and has been used for strong basis the new chimeric promoters because of express special to smooth muscle cell.The invention also discloses the carrier that contains this promotor and they in vivo, exsomatize and external application in the cell transfer gene.Construct of the present invention allows opposite characteristic is combined for the first time, i.e. highly selective and high transcriptional activity.Therefore, the invention provides a kind of interior or external especially effectively means of expressing and regulating this expression at the smooth muscle cell directed gene of body that are used for.
The present invention more specifically is to have made up chimeric (heterozygosis) promotor in the zone of containing different sources and function.More particularly, first purpose of the present invention is a kind of hybrid promoter, and it comprises:
The enhancing subarea of-a kind of omnipresence type strong promoter/enhanser all or part of and
-allow promoter region specific expressed in smooth muscle cell.
Reported the associating that strengthens subarea and promoter region in the prior art.For example, known with CMV the enhancing subarea and the promotor coupling (WO96/13597) of non-specific promotor such as chicken β actin gene to improve their effectiveness.But do not describe or hint that this construct is to be used for attempting obtaining the purpose expressed at the smooth muscle cell high specificity.The present invention part is based on to specific " enhanser " element and specific " promotor " selection of components and combination.The present invention has been also based on having proved that this combination of elements can obtain high-caliber expression, and do not influence the selectivity of promotor to smooth muscle cell.Thereby the present invention provides particularly advantageous construct, because they allow some molecules of directed high level production in smooth muscle cell.In addition, the application proves that also these constructs can be used for externally also can be used in the body.
In hybrid promoter of the present invention, strengthen the subarea and be connected with promoter area function, promptly make the performance of enhanser district to the active hormesis of promoter region.General these two zones connect in the genetic engineering mode, and mutually enough near activating promoter region so that strengthen the subarea.Preferably, the spacing between enhancing subarea and promoter region is more preferably less than 500bp less than 1kb.In the particularly preferred construct of the present invention, the spacing in these two zones is less than 400bp, preferably less than 200bp.In addition, as shown in embodiment, the activity of two zone not remarkably influenced of direction hybrid promoters of the present invention separately.Therefore, strengthening the subarea can be with the identical or opposite direction location of relative promoter region transcriptional orientation.
In a preferred embodiment, strengthen the subarea and be selected from the enhancing subarea of cytomegalovirus immediate early gene (CMV-IE), the enhancing subarea of Rous sarcoma virus LTR (LTR-RSV), the enhancing subarea of SV40 virus, the enhancing subarea of EF1 α gene.More preferably, in hybrid promoter of the present invention, strengthening the subarea is the enhancing subarea of cytomegalovirus immediate early gene (CMV-IE), (hCMV-IE) of preferred human cytomegalic inclusion disease virus.Specific enhancing subarea for example is made of-522 to-63 fragments of hCMV-IE gene, or constitutes by containing this district's at least a portion and having the active any fragment of enhanser.
As for promoter region,, enforcement the present invention comprises smooth muscle cell α-Ji Dongdanbai encoding gene (SMact) promotor or all or part of zone of SM22 gene promoter for advantageously utilizing.The promotor of these genes (is seen Ueyama H. etc., Mol.Cell.Biol., 4 (1984) 1073-1078 especially to the existing report of the specificity of smooth muscle cell; SolwayJ. etc., J.Biol.Chem., 270 (1995) 13460-13469).
First particularly advantageous scheme of the present invention is made of following hybrid promoter, and it comprises:
-human cytomegalic inclusion disease virus immediate early gene (hCMV-IE) strengthens all or part of of subarea, and
The promotor of people Smact gene-smooth muscle cell α-Ji Dongdanbai (SMact) encoding gene, preferred is all or part of.
Second particularly advantageous scheme of the present invention is made of following hybrid promoter, and it comprises:
-human cytomegalic inclusion disease virus immediate early gene (hCMV-IE) strengthens all or part of of subarea, and
The promotor of mouse SM22 α gene-SM22 gene, preferred is all or part of.
In addition, in a specific embodiments of the present invention, used promoter region is to contain basic promotor and the chimeric district that brings the sequence of tissue specificity, and described sequence is from SMact promotor or SM22 promotor, or from both.In this embodiment, basic promotor can be " minimum " promotor, promptly only contains the active essential sequence (as the TATA box) of promoter transcription.This basis promotor can be the basic promotor itself of SMact or SM22 gene, or allogenic (as betaglobulin, HSV-TK, SV40 or EF1-α).Bring tissue-specific sequence advantageously to comprise SMact promoter sequence (R.T.Shimizu etc., J.Biol.Chem.270 (1995) 7634-7643) and/or SM22 promotor (L.Li etc., J.Cell.Biol.132 (1996) 849-859; S.Kim etc., J.Clin.Invest.100 (1997) 1006-1014; Kemp etc., Bochen.J.310 (1995) 1037-1043) a part.
A kind of particular type of hybrid promoter of the present invention comprises:
The enhancing subarea of-omnipresence type strong promoter/enhanser is all or part of,
-basic promotor and
-contain SMact and/or SM22 promotor all or part of bring tissue-specific sequence.
In order to make up hybrid promoter of the present invention, can utilize Protocols in Molecular Biology well known by persons skilled in the art.For example, strengthen the subarea and can separate from nucleic acid library or from full cell DNA according to a conventional method with promoter region (comprise basic promotor and bring tissue-specific sequence) and obtain, for example by increasing with specific probe.These fragments can also utilize that existing sequence information carries out synthetic in the prior art.After obtaining these fragments, can utilize ligase enzyme or other restriction enzymes to be easy between it, connect, to produce hybrid promoter of the present invention.In addition, these fragments can also be through digestion, sudden change, insert or add base pair and be modified, so that its clone or change its functional performance.In addition, as previously mentioned, can directly connect between these fragments, or by the active base pair of not remarkably influenced hybrid promoter at interval.
Hybrid promoter of the present invention has the ability of specific expressed purpose nucleic acid in smooth muscle cell." special " property of expressing refers to that the promoter activity in smooth muscle cell is significantly high.Though may there be non-specific expression in other cells, corresponding activity level is compared generally very low (can ignore) with observed level in smooth muscle cell, general low at least 10 times.The result who provides in an embodiment shows that differential expression can reach 140 times, and this has proved the strong selectivity of promotor of the present invention.To this, the result who provides also shows the high specific for smooth muscle cell, because do not detect any expression in the epithelial cell that is adjacent in blood vessel, especially artery.The result that embodiment provides also proves the effectiveness of the effectiveness of promotor of the present invention far above the specificity promoter of non-heterozygosis, and difference can reach more than 100 times.Therefore, these theory of factors understand that hybrid promoter of the present invention is for expressing target nucleic acid in the beat all advantageous feature aspect effectiveness and the specificity in smooth muscle cell.
To this, another target of the present invention relates to and contains the coding target RNA that is under the control of above-mentioned hybrid promoter or the expression of nucleic acids box of polypeptide.Advantageously, expression cassette of the present invention also contains the transcription termination signal that is in nucleic acid 3 ' end.
Consider expression cassette of the present invention directed cell mass, described nucleic acid codified for example is selected from following albumen:
-cell cycle associated protein is as p21 or any other cell cycle protein dependent kinase (cdk) repressible protein, the product of retinoblastoma (Rb) gene, GAX, GAS-1, GAS-3, GAS-6, Gddd45, Gadd153, cyclin A, B and D.
The albumen of-cell death inducing, as p53, the member of apoptosis-inducing protein family such as Bas, Bcl-X
6, Bad or Bcl
2And Bcl-X
1Any its antagonist.
-can change the albumen of smooth muscle cell proliferation, as the intrabody or the ScFv of the protein-active that suppress to participate in cell proliferation, Ras albumen for example, map kinases, or tyrosine kinase receptor or growth factor receptors.
-be used to carry out the marker protein (LacZ, GFP, Luc, secretor type alkaline phosphatase (SeAP), tethelin (GH) etc.) of proliferation research or diagnosis research.
The albumen that-induction of vascular generates, member as VEGF family, the member of FGF family, particularly FGF1, FGF2, FGF4, FGF5, angiogenin, EGF, TGF α, TGF β, TNF α, the Scatter factor/HGF, the member of vascularization albumen (angiopoetine) family, particularly comprises the interleukin-of IL-1, IL-2, IL-8, angiotonin-2 at cytokine, profibr(in)olysin activation factor (TPA), urokinase (uPA), participate in active fat synthetic molecule (prostaglandin(PG), Cox-1).
-transcription factor, for example contain the natural or chimeric nuclear receptor of DNA in conjunction with territory, ligand binding domain and transcription activating or inhibition territory, as fusion rotein tetR-NLS-VP16, fusion rotein from estrogen receptor, fusion rotein from steroid hormone receptor, fusion rotein from progesterone receptor, albumen (the Rivera etc. (1996) of the CID system (chemical inducer of dimerization) that Rivera etc. describe, the humanization system of genetic expression medicine control, nature medical science (Nature Modecine), 2:1028-1032).
Should be understood that the specific examples that the invention is not restricted to these albumen or RNA, those skilled in the art can utilize the present invention to operate in by simple normal experiment and express any nucleic acid in the smooth muscle cell.
Another object of the present invention also relates to any carrier that contains above-mentioned hybrid promoter or expression cassette.Carrier of the present invention for example can be plasmid, clay or any not by the DNA of viral capsidization, phage, artificial chromosome, recombinant virus etc., preferred matter or recombinant virus.
In the plasmid-type carrier, can enumerate clone and/or the expression plasmid that generally has replication orgin well known by persons skilled in the art.Can also mention the freshly prepd plasmid that for example has by improved replication orgin described in patent application WO96/26270 and the PCT/FR96/01414 and/or mark.
In recombinant virus type carrier, preferred recombinant adenovirus, retrovirus, simplexvirus or adeno associated virus.The structure of such replication defective recombinant virus has a large amount of reports in the literature, these viral infection characterizations also are so (to see S.Baeck and K.L.March (1998) especially, Circul.Research vol.82, pp295-305), T.Shenk, B.N.Fields, D.M.Knipe, P.M.Howley etc. (1996), Adenoviridae: virus and its duplicate (virusology) (Adenoviridae:the viruses andtheir replication (in virology)), pp211-2148, EDS-Ravenspublishers/Philadelphia, P.Yeh﹠amp; M.Perricaudet (1997), FASEB Vol.11, pp615-623.
Being used to implement particularly preferred recombinant virus of the present invention is defect type recombination adenovirus.
Adenovirus is the linear dsdna virus that is about 36kb (kilobase), has different serotype, and its structure is slightly different with characteristic, but has similar gene structure.More particularly, recombinant adenovirus can be from the human or animal.For the adenovirus in people source, preferably be included into those of C group, particularly 2 types (Ad2), 5 types (Ad5), 7 types (Ad7) or 12 types (Ad12) adenovirus.In the various adenovirus of animal-origin, all strains [for example manhattan strain or A26/61 strain (ATCC VR-800)] of the adenovirus, particularly CAV2 adenovirus in preferred dog source.Other adenovirus of animal-origin have been enumerated especially at patent application WO94/26914 (being incorporated herein by reference).
The adenoviral gene group comprises each terminal inverted repeats (ITR), capsidation sequence (Psi), early gene and late gene especially.Known main early gene is in E1, E2, E3 and the E4 district.Wherein, known gene is essential to viral proliferation especially in the E1 district.Known main late gene is in L1 to the L5 district.The genome of Ad5 adenovirus checks order fully and can obtain (seeing Genebank M73260 especially) in the data preface.The part of other adenoviral gene group (Ad2, Ad7, Ad12 etc.) even all also checked order.
For as recombinant vectors, prepared different constructs from adenovirus, it contains various therapeutic genes.In every kind of these construct, adenovirus is modified, and it can not be duplicated in infected cell.Therefore, construct described in the prior be the E1 district (for virus replication institute must) lack and inserted at this position exogenous DNA array adenovirus (Levrero etc., Gene 101 (1991) 195; Gosh-Choudhury etc., Gene 50 (1986) 161).In addition, in order to improve the characteristic of carrier, have been proposed in and set up other disappearances or modification in the adenoviral gene group.For example, in the ts125 variant, introduced a thermostability point mutation, the inactivation (Van der Vliet etc., 1975) that makes that the DNA of 72kDa is conjugated protein (DBP).Other carriers are in the E4 district disappearance to be arranged at another virus replication and/or breeding required area.The E4 district participate in late gene expression adjusting, late period nRNA stability, the expression of eliminating host cell proteins and the efficient of viral dna replication.Therefore the back ground noise of the virogene transcript and expression of the adenovirus carrier that all lacks of E1 and E4 district is very little.For example among patent application WO94/28152, WO95/02697, the WO96/22378 such carrier is being described.In addition, on the IVa2 gene, there is the carrier of modification that report (WO96/10088) is also arranged.
In a preferred embodiment of the invention, recombinant adenovirus is C group adenovirus hominis, more preferably adenovirus Ad2 or Ad5.
The used recombinant adenovirus of the present invention is preferably in the disappearance that has in its genome in the E1 district, more preferably lacks E1a and E1b district.As a concrete example, can lack 454-3328,382-3446 or 357-4020 Nucleotide (with reference to the Ad5 genome).
According to a kind of preferred version, the used recombinant adenovirus of the present invention also has the disappearance in the E4 district in its genome.Disappearance in preferred this E4 district influences whole open reading frame.For example concrete disappearance can be 33466-35535 or 33093-35535.Ask other disappearances of having described among WO95/02697 and the WO96/22378 (being incorporated herein by reference) in the E4 district in the patent.
Expression cassette can be inserted on the different loci of recombination group, can be inserted in E1, E3 or the E4 district, replaces deletion sequence or is added on wherein.Also can be inserted in any other site outside the essential sequence (ITR sequence and capsidation sequence) of virus production cis.
In capsidation clone, produce recombinant adenovirus, clone that promptly can the one or more defective functions of trans additional recombinant adenovirus genome.In capsidation clone well known by persons skilled in the art, for example can mention 293 clones, wherein integrated the part of adenoviral gene group.More particularly, 293 clones are people's tire kidney cell lines, it contains adenoviral serotype 5 (Ad5) genome left end (about 11-12%), comprises left side ITR, capsidation district, E1 district (comprising E1a and E1b), the proteic coding region of pIX and the proteic part of pIVa2 coding region.This clone can trans additional E1 district defective recombinant adenovirus (it is all or part of promptly to lack the E1 district), and produce the infectious titer original seed.This clone can also be produced the viral original seed that contains thermostability E2 sudden change in addition down in the temperature (32 ℃) that allows.The some other clone that can replenish the E1 district also has report, particularly based on human lung cancer cell A549 (WO94/28152) or based on (Hum.Gen.Ther. (1996) 215) of human retina's cell.Also reporting in addition can trans clone of replenishing multiple adenovirus function.Specifically can enumerate the clone (Yeh etc., J.Viol.Vol.70 (1996), the pp559-565 that replenish E1 and E4 district; Cancer Gen.Ther.2 (1995) 322; Krougliak etc., Hum.Gen.Ther.6 (1995) 1575) and the clone of additional E1 and E2 (WO94/28152, WO95/02697, WO95/27071).
Generally by introduce viral DNA in capsidation system, (the adenovirus kinetics cycle is 24-36 hour) dissolved cell produces recombinant adenovirus behind about then 2-3.In order to implement this method, the viral DNA of introducing can be transfection in cell, randomly bacterium (WO96/25506) or in enzyme mother (WO95/03400), make up, recombinant virus genomes completely.It can also be the recombinant virus that is used to infect capsidation clone.Viral DNA can also be introduced with the pieces that respectively has a recombinant virus genomes part and a homologous region, and the back rebuilds this recombinant virus genomes by the homologous recombination between different fragments in introducing the capsidation cell.
After the cytolysis, separate recombinant virus particle by caesium chloride density gradient centrifugation.Among the patent application FR96:08164 (being incorporated herein by reference) another kind of method has been described.
The invention still further relates to the composition that contains above-mentioned carrier and chemistry or biochemical transfering reagent.Term " chemistry or biological transfering reagent " refers to help any compound (promptly not being recombinant virus) that nucleic acid penetrates into cell.This can right and wrong virus cationoid reagent such as cationic lipid, peptide, polymkeric substance (polymine, polylysine), nano particle; Or the non-viral reagent of non-cationic such as non-cationic liposome, non-cationic polymkeric substance or nano particle.This reagent is well known by persons skilled in the art.
The invention still further relates to the composition that the recombinant virus that contains as defined above and physiology can be accepted carrier.
The invention still further relates to and contain the pharmaceutical composition of carrier as mentioned above.Pharmaceutical composition of the present invention can be mixed be suitable in epidermis, oral, parenteral, the nose, the form of administrations such as vein, intramuscular, subcutaneous, intraocular, transdermal.
Preferably, pharmaceutical composition of the present invention contains the pharmaceutically acceptable carrier that is useful on injection formulations, particularly intravenous injection or smooth muscle tissue's injection liquid, sterile isotonic salt solution (monosodium phosphate, disodium salt, sodium-chlor, potassium, calcium or magnesium etc. particularly, or the mixture of these salt), or be dry composition, dried frozen aquatic products particularly, it can be configured to injection liquid after adding sterilized water or physiological saline according to circumstances.Also can use other vehicle such as hydrogel.This hydrogel can be made with any biocompatibility nontoxic polymer (homopolymerization or assorted poly-).These polymkeric substance were for example disclosing among the patent application WO93/08845.Wherein there are some to buy, particularly those that obtain from ethylene oxide and/or propylene oxide.The application of hydrogel is in the vessel wall, particularly the nucleic acid in the smooth muscle cell of vessel wall shifts particularly advantageous.Injected dose can be according to various parameter regulation, particularly according to used administering mode, the target of being pursued (mark, pathology detect etc.), gene to be expressed or require the time length expressed.Generally speaking, recombinant virus of the present invention is with 10
4To 10
14Pfu, preferred 10
6To 10
10Dosage form preparation and the administration of pfu.Pfu (plaque forming unit) is equivalent to the infection ability of viral solution, is to determine by the plaque number that infects suitable cell culture and measure infected cell.Reported the determination techniques of viral solution pfu titre in the document in a large number.For external or stripped application, expression cassette of the present invention, carrier or composition can be incubated in the presence of selected cell mass with routine dose.This insulation can be carried out in incubator, flask, fermentor tank or selected any other device.
In addition, the invention still further relates to any cell of transforming by expression cassette or carrier (particularly adenovirus) as mentioned above.The cell of " transformation " refers to contain the cell of construct of the present invention.But these cell produced in vitro recombinant proteins.These cells also can be used in the implantable bioartificial body, as pressing method described in the patent application WO95/14785.These cells are human smooth muscular cells preferably.
The invention still further relates to as mentioned above, hybrid promoter is used for application external, stripped, the specific expressed nucleic acid of the inherent smooth muscle cell of body.
The invention still further relates to hybrid promoter as defined above is used at smooth muscle cell and the not application in the composition of express nucleic acid in the adjacent epithelial cell in artery in preparation.
Because its specificity to smooth muscle cell, construct of the present invention also can be used for creating the animal model of vascular disease or is used for carrying out marker research or is used for the detection or the method for detecting of sample smooth muscle cell.
The invention still further relates to the production method of recombinant protein, be included in and introduce above-mentioned carrier in the cell mass, cultivate described reconstitution cell group and reclaim the described albumen that produces.In order to implement method of the present invention, it is favourable using smooth muscle cell, and this can be clone or the primary culture of having set up.
To describe the present invention in more detail by means of the following examples, these embodiment should be considered as illustrative and nonrestrictive.
Description of drawings
Table I: the relative reactivity of the hybrid promoter (hSM α-Ji Dongdanbai) that external temporary transient transfection is measured in rabbit smooth muscle cell primary culture (rabbit SMC), ECV304 cell, C2C12 sarcoplast, HeLa cell, NIH3T3 cell, TU182 cancer cells and 293 nephrocytes.The relative reactivity of each promotor is represented with the per-cent of relative pCMV-leadTK plasmid gained luciferase activity.The enhancer sequence of Enh-X:hCMV-IE is cloned in the upstream of X promotor with its normal direction.The enhancer sequence of Hne-X:hCMV-IE is cloned in the upstream of promotor X in the opposite direction.
Table II: the relative reactivity of the hybrid promoter (mSM22) that external temporary transient transfection is measured in rabbit smooth muscle cell primary culture (rabbit SMC), ECV304 cell, C2C12 sarcoplast, HeLa cell, NIH 3T3 cell, TU182 cancer cells and 293 nephrocytes.The relative reactivity of each promotor is represented with the per-cent of the luciferase activity that relative pCMV-leadTK plasmid obtains.The enhancer sequence of Enh-X:hCMV-IE is cloned in the upstream of promotor X with its normal direction.The enhancer sequence of HnE-X:hCMV-IE is cloned in the upstream of promotor X in the opposite direction.
Fig. 1: the plasmid synoptic diagram, wherein expression cassette contains hSM α-Ji Dongdanbai hybrid promoter.
Fig. 2: the plasmid synoptic diagram, wherein expression cassette contains mSM22 α hybrid promoter.
Fig. 3: in rabbit smooth muscle cell primary culture (rabbit SMC), epithelial cell (ECV304), mouse muscle-forming cell (C2C12) and the hybrid promoter activity that external temporary transient transfection is measured from human cervical carcinoma's epithelial cell (HeLa) from people's umbilical cord cancer.The relative reactivity of each promotor is represented with the per-cent of relative pCMV-leadTK plasmid gained luciferase activity.The enhancer sequence of Enh-X:hCMV-IE is cloned in the upstream of X promotor with its normal direction.The enhancer sequence of Hne-X:hCMV-IE is cloned in the upstream of promotor X in the opposite direction.
Fig. 4: the activity of the hybrid promoter that external temporary transient transfection is measured at little mouse embryo desmocyte (NIH 3T3), from the cell (TU182) of people ORL cancer and people's tire nephrocyte (293) of transforming.The relative reactivity of each promotor is represented with the per-cent of the luciferase activity that relative pCMV-leadTK plasmid obtains.The enhancer sequence of Enh-X:hCMV-IE is cloned in the upstream of promotor X with its normal direction.The enhancer sequence of HnE-X:hCMV-IE is cloned in the upstream of promotor X in the opposite direction.
Fig. 5: the activity of the hybrid promoter that vivo gene transfer is estimated in the tibialis before the C57BL6 mouse.The relative reactivity of every kind of promotor is represented with the per-cent of the luciferase activity that relative pCMV-leadTK plasmid obtains.
Material and method
In the molecular biology in the electroelution purifying of the caesium chloride density gradient centrifugation of the preparation extraction of used ordinary method such as plasmid DNA, plasmid DNA, agarose gel electrophoresis, dna fragmentation, the salt solution plasmid DNA be well known by persons skilled in the art with transforming in ethanol or isopropanol precipitating, the intestinal bacteria, a large amount of description (Sambrook etc. are arranged in the literature, " Molecular Cloning; a Laboratory Mannual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
The plasmid pGL3-Basic that is used to clone various promoter regions is commercial source (Promega Corporation).Plasmid pCMV β (Clontech Laboratori esInc.) and pUC18 (Boehringer Mannheim) also are commercial source.
The ACP method enzymatic amplification of dna fragmentation (polymerase chain amplification) can utilize DNA thermal cycler (Perkin Elmer Letus) to be undertaken by the explanation of manufacturers.
The intestinal bacteria electroporation of plasmid DNA can utilize electroporation device (Bio-Rad) to be undertaken by the explanation of manufacturers.
The checking of nucleotide sequence can illustrate by manufacturer by the test kit that the method (Proc.Natl.Acad.Sci.USA, 74 (1977) 5463-5467) of Sanger etc. utilizes Applied Biosystems to provide to be carried out.
Embodiment
Embodiment 1: hybrid promoter and contain the structure of their expression plasmid.
1.1 hybrid promoter hSM α-Ji Dongdanbai plasmid phSMact. presses (Sambrook etc. such as Sambrook, " MolecularCloning; a Laboratory Mannual ", Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989) described method has prepared the high-molecular weight genomic dna from human aortic smooth muscle cell's primary culture (Clonetics).
This DNA carries out the ACP amplification first time as template with following primer:
-primer 6417 (5 ' GATGGTCCCTACTTATGCTGCTA 3 ') (SEQ ID 1) is from-1034 (promoter region) (UeyamaH. etc., Mol.Cell.Biol., 4 (1984) 1073-1078 of the special unstriated muscle α-Ji Dongdanbai of people gene.Genbank D00618).
-primer 6418 (5 ' CTTCCATCATACCAAACTACATA 3 ') (SEQ ID 2), 1974 of sequence D 00618 are positioned within first intron of hSMact gene.Reaction mixture comprises 1mg genomic dna, every kind of primer of 10pmol (6417 and 6418), the various deoxyribonucleotides (dATP, dCTP, dGTP, dTTP) of 100mM, 2mM MgCl
2With the 5 Taq DNA of unit polymkeric substance (Perkin Elmer).Reaction volume is mended to 50ml with the ACP damping fluid that Perkin Elmer recommends, and transfers to optimum concn.
The ACP amplification is at Micoamp
TMUtilize thermal cycler PTC-100 in the test tube
TM(MJResearch Inc.) carries out.This amplification comprises step 95 ℃ of sex change in 2 minutes, is 30 circulations then: 15 seconds, 60 ℃ hybridization of 95 ℃ of sex change were extended 1 minute in 30 seconds and 72 ℃.Be extra the extension 5 minutes after these 30 circulations, then the ACP reaction solution remained on 10 ℃.
From this first reaction, get 1 microlitre reaction solution, with the dilution of 10ml water.This diluent of 1ml is used to carry out the ACP second time then, and condition is identical with for the first time (above-mentioned), but primer is to difference:
-primer 6453 (5 ' CTGCTAAATTGctcgagGACAAATTAGACAAA 3 ') (SEQ ID 3), this primer is introduced an XhoI site (lowercase of underscore) promotor hSMact upstream (680).
-primer 6456 (5 ' CCCTGACAaagcttGGCTGGGCTGCTCCACTGG 3 ') (SEQ ID 4), this primer hSMact+30 introduce a HindIII site.
Behind analysis, purifying on the sepharose, the dna fragmentation of ACP amplification digested 3 hours in 37 ℃ with XhoI and HindIII, be cloned in then in advance among the carrier pGL3-Basic (Promega) with these identical restriction enzymes digestion, make plasmid phSMact (Fig. 1).Plasmid pXL3130 and pXL3131. are included in between replication orgin meter-522 and-63 corresponding to the dna fragmentation in the enhancing subarea of human cytomegalic inclusion disease virus IE gene (hCMV-IE) promotor, as primer it are increased through ACP as template with oligonucleotide 8557 (5 ' ATC GAC GCGTGC CCG TTA CAT AAC TTA CGG 3 ') (SEQ ID 5) and 8558 (5 ' ATCGAC GCG TCC GCT CGA GCG TCA ATG GGG CGG AGT TG 3 ') (SEQ ID6) with plasmid pCMV β.This fragment digests with MluI, is cloned in then in advance with among MluI digestion and the plasmid phSMact with alkaline phosphatase treatment.According to segmental direction of insertion difference, two different plasmids: pXL3130 and pXL3131 have been obtained.The synoptic diagram of these plasmids (Fig. 1) shown in the drawings.These plasmids contain the hybrid promoter of MluI-NcoI pieces, and it is made of the enhanser of hCMV-IE gene promoter and the promotor of hSM α-Ji Dongdanbai gene.
1.2 hybrid promoter mSM22
Plasmid pmSM22. presses currently known methods (Sambrook etc., " MolecularCloning, a Laboratory Mannual ", Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989) from the liver of Balbc mouse, prepared the high-molecular weight genomic dna.
Carry out the ACP amplification with this DNA as template and following primer:
-primer 6517:
(5 ' CCAGGCTGCActcgagACTAGTTCCCACCAACTCGA 3 ') (SEQ ID7), this primer is at mouse SM22 α gene promoter (Solway J. etc., J.Biol.Chem., 270 (1995) 13460-13469; Genbank number: introduce XhoI site (lowercase of underscore) for L41161)-436.
-primer 6518:
(5 ' TCGTTTGaagcttGGAAGGAGAGTAGCTTCGGTGTC 3 ') (SEQ ID8), this primer mSM22 α+43 draw the XHindIII site.
With with used identical reagent of hSMact and identical prepared at concentrations contain the reaction mixture of 1mg mouse gene group DNA and the various primers of 10pmol (6517 and 6518), carry out ACP amplification (seeing embodiment 1.1) then under the same conditions.
Analyze on the sepharose and purifying after, the dna fragmentation of this ACP amplification has been digested 3 hours for 37 ℃ with XhoI and HindIII, be cloned in then in advance among the carrier pGL3-Basic (Promega) that digests with identical restriction enzyme.The plasmid that forms is called pmSM22 (Fig. 2).
Plasmid pXL3152 and pXL3153. are contained in respect between transcripting start point-522 and-63 corresponding to the enhancing subarea of hCMV-IE gene promoter, with plasmid pCMV β as template, as primer it is carried out the ACP amplification with oligonucleotide 8557 and 8558: 8557 (5 ' ATC GAC GCG TGC CCG TTA CAT AAC TTA CGG 3 ') (SEQID 5); 8558 (5 ' ATC GAC GCG TCC GCT CGA GCG TCA ATG GGGCGG AGT TG 3 ') (SEQ ID 6).This fragment digests with MluI, is cloned in then in advance with among MluI digestion and the plasmid pmSM22 with alkaline phosphatase treatment.According to this segmental direction of insertion difference, two different plasmids: pXL3152 and pXL3153 have been obtained.Illustrating in Fig. 2 of these plasmids.These plasmids contain the hybrid promoter of MluI-NcoI pieces, and it is made of the enhanser of hCMV-IE gene promoter and the promotor of mSM22 gene.
1.3 control plasmid pCMV-leadTK
By (Cell such as Tanaka, 60 (1990) 375-386) Bao Dao expression vector pCGN contains that (+CMV the promotor that 51/+101) merges (522/+72), is positioned at the upstream of the encoding sequence of hemagglutinin epi-position with " leader " (leader sequence) of HSV tk gene.Carry out the ACP amplification with plasmid pCGN (10ng) as template.With hSMact and the used identical condition of nSM22 under (embodiment 1.1 and 1. 2) carry out ACP reaction and amplification.The primer is as follows:
-primer 6718 (5 ' CCCGTTACATAACTTACGGTAAATGGCCCG 3 ') (SEQID9), the hybridization of the CMV promotor at 8 Nucleotide places-522 of this primer and downstream, pCGN EcoRI site.
-primer 6719 (5 ' gGGACGCGCTTCTACAAGGCGCTGGCCGAA 3 ') (SEQID 10), this primer hybridization is to 101 of tk " leader ".This point hereinafter will be explained in the NcoI site that first black matrix Nucleotide G is used to repair pGL3-Basic.
With the ACP fragment purification that so obtains, use T4 phage polynucleotide kinase (NewEngland Biolabs) phosphorylation then.Abreast, carrier pGL3-Basic (Promega) uses Klenow archaeal dna polymerase (BoehringerManhein) to handle, to fill and lead up the NcoI site with NcoI linearizing, purifying then.This carrier is used alkaline phosphatase (Boehringer Manheim) dephosphorylation subsequently, is used for the segmental insertion of ACP of phosphorylation then.Like this, have only when CMV-leader tk is segmental and be orientated 5 ' (primer 6718 partly,-522 of CMV) be positioned at downstream, pGL3-Basic HindIII site and its 3 ' end (primer 6719, leader tk) with the NcoI site (first ATG of luciferase) of pGL3-Basic when being connected, the black glycosides (G) of primer 6719 is repaired the NcoI site.So the plasmid that obtains is called as pCMV-leadTK.
Embodiment 2: the external specificity of hybrid promoter
The vitro tissue specificity characteristic of present embodiment explanation hybrid promoter of the present invention.
2.1 cell cultures
The smooth muscle cell of rabbit (SMC) is cultivated the DMEM that 20% foetal calf serum (SVF) arranged in benefit
TMIn the substratum (Life Technologies Inc).The ECV304 cell cultures has 199 of 10%SVF in benefit
TMIn the substratum (Life Technologies Inc.).Sarcoplast C2C12, HeLa cell, NIH 3T3 cell and TU182 cell cultures have the DMEM of 10%SVF in benefit
TMIn the substratum.293 cell cultures have the MEM of pyruvic acid, non-essential amino acid and 10%SVF in benefit
TMIn the substratum (Life Technologies Inc).All are cultivated in 37 ℃ of incubators at atmospheric moisture and 5%CO
2Divide to depress and carry out.
2.2 in-vitro transfection
Carry out transfection in 24 orifice plates, each transfection is carried out 3 times.In transfection preceding 24 hours, inoculating cell: (1) rabbit smooth muscle cell, ECV304, NIH3T3 and HeLa cell, every hole 5x10
4Individual cell, (ii) TU182 cell, every hole 10
5Individual cell, (iii) C2C12 cell, every hole 3x10
4Cell reaches (iv) 293 cells, every hole 2 * 10
5Individual cell.
In every hole, 500ng plasmid DNA (250ng target plasmid and 250ng pUC18) and cationic lipid RPR120535B (WO97/18185) with the mixed of 6nmol fat/μ g DNA in the DMEM that contains 150mM NaCl and 50mM supercarbonate
TMIn the substratum (final volume 20 μ l).After following 20 minutes of the room temperature, in the presence of no SVF with 20 μ l DNA/ lipoprotein mixtures and cells contacting 2 hours.In substratum, replenish SVF then, to reach every kind of desired percentage ratio of cell cultures.
After the transfection 48 hours, take out substratum, cell is with PBS (Life TechnologiesInc.) washing 2 times.Use Luciferase Assay System then
TM(luciferase assay system) test kit (Promega Corporation) is measured luciferase activity by the explanation of manufacturer.
2.3 the specific activity of hybrid promoter
In 7 kinds of different cells the relative luciferase activity of the hybrid promoter of external test (with respect to the pCMV-headTK promotor) be summarised in Fig. 3 and 4 and Table I and II in.The result shows, the relative reactivity value of hSMact (Table I) and mSM22 (Table II) promotor clearly illustrates its specificity for smooth muscle cell, this specific specificity existing in the literature report (Skalli etc., J.Histochem.Cytochem., 37 (1989) 315-321; Shimizu etc., J.Biol.Chem., 270 (1995) 7631-7643; Li etc., J.CellBiol., 132 (1996) 849-859).In fact, the relative reactivity in rabbit SMC is at least 5 times of observed level in the other types cell: (i) the hSMact promotor is 5-20 doubly (Table I); (ii) the mSM22 promotor is 5-25 doubly (Table II).
The result clearly illustrates that shown in Table I and the II, and in smooth muscle cell, the activity of 4 hybrid promoters of the present invention (Enh-hSMact, hnE-hSMact, Enh-mSM22 and hnE-mSM22) is suitable with the CMV promotor on intensity.And every kind of these promotors relative activity ratio in the other types cell observed level low at least 10 times (for hSMact hybrid promoter) and at least 4 times (for mSM22 promotor) in smooth muscle cell: being 10-140 times to the hSMact hybrid promoter (i), is 4-55 times to the mSM22 hybrid promoter (ii).Thereby these hybrid promoters have kept the tissue specificity identical with mSM22 with specificity promoter hSMact.
These results also show in addition, strengthen its activity of not remarkably influenced of direction in subarea in the hybrid promoter of the present invention.
Thereby these 4 hybrid promoters have the same strong activity with CMV promotor (being celebrated with strong promoter) in external smooth muscle cell, have kept the tissue specificity suitable with the mSM22 promotor even higher with hSMact simultaneously.
Embodiment 3: the body internal specific of hybrid promoter
The in-vivo tissue specificity characteristic of present embodiment proof hybrid promoter of the present invention.
3.1 the transgenosis in the skeletal muscle
In the preceding tibialis with different plasmid intramuscularly to 5 female C57BL6 mouse in age in week.Every kind of plasmid is diluted in the NaCl solution of 150mM (final concentration), with the amount injection of every muscle 10 μ g.Injected back 3 days, the muscle sampling is placed on 2ml Cell Cultura lysisReagent
TMIn (cell culture solubilising reagent) damping fluid (Promega Corporation), pulverize centrifugal 15 minutes then with 4000g with Diax homogenizer (Heioloph).Use Luliferase Assay System then
TMTest kit (Promega Corporation) is measured luciferase activity by manufacturer's explanation.
3.2 the activity in vivo of hybrid promoter in skeletal muscle
The relative reactivity of also before mouse, shifted interior evaluating behind the naked DNA in the tibialis two specificity promoters (hSMact and mSM22) and 2 hybrid promoters of the present invention.Result shown in Fig. 5 shows that the activity of Enh-hSMact promotor is 100 times of CMV promoter activity.Equally, the activity of Enh-mSM22 promotor is 17 times of CMV promoter activity.Therefore, observe external viewed tissue specificity in vivo, especially seen in the C2C12 cell, the latter be with body in used immediate model.
Embodiment 4: make up specific hybrid promoters control and express the proteic recombinant adenovirus of GAX down
The purpose of present embodiment is to describe a kind of adenovirus carrier, and it has the proteic encoding gene of the GAX that can be operatively connected with hybrid promoter of the present invention, and described promotor constitutes (enh-hSMact) by cmv enhancer and SM α-Ji Dongdanbai promotor.
People gax gene comprises 912 base pairs, and coding participates in cell cessation of growth cessation (growth-arrest-specific homeobox, cessation of growth cessation specificity homology frame) and to effective 303 the amino acid whose transcription factors of the propagation of human smooth muscular cells.This homeodomain gene initial separation is from aorta, and expression (Gorski etc., 1993) in adult's cardiovascular organization especially.
The sequence of people gax gene has been used from the sequence of people gax gene to come out from the cDNA library clone of skeletal muscle through PCR (polymerase chain reaction) as primer, and by people such as Walsh announce (Genomics (1994), 24, p535).Then with this sequence clone in expression vector pXL3297.This plasmid is from plasmid Bluescript (Stratagene), contain people CMV IE enhancers/promoters (522/+72) (and Cell (1985), 41, p521) and the polyA (2538-2759) (Genbank position SV40CG) of SV40.
Use the plasmid pXL3130 described in the embodiment 1 (Fig. 1) to make up, introduce SM α-Ji Dongdanbai promotor in the described plasmid in advance.Plasmid pXL3297 contains people gax expression carrier.It is digested so that people gax gene is introduced among the same aforementioned plasmid pXL3282 with HindIII and AvrII enzymic digestion with HindIII and AvrII, obtain plasmid pXL3300.As shown in Figure 6, wherein describe each step of above-mentioned plasmid construction in detail, final plasmid pXL3310 contains an expression cassette, its by the CMV-IE enhanser that connects by the present invention and SM α-Ji Dongdanbai promotor (pSMA) and with the termination signal polyA formation of its people GAX protein coding gene that can be operatively connected and SV40.
With plasmid and adenovirus in capsidation cell cotransfection and the homologous recombination of this people gax expression casette, be introduced in the serotype 5 recombinant human adenovirus (Ad5) that lacked E1 and E3 district then by having expression cassette.Infect after 2 or 3 days dissolving capsidation cell and in the cesium chloride gradient centrifugal recombinant virus particle, obtain containing the adenovirus original seed of people gax expression casette.
Then virion is used for studying the expression at smooth muscle cell under the control of promotor of the present invention of people gax gene.
Carry out immunofluorescence or Western trace with the anti-gax polyclonal antibody of rabbit and infecting the proteic expression of 24 hours posteriority proof GAX of unstriated muscle primary cell.
Carry out the expression of dot blotting and Northern trace 24 hours analysis messenger RNA(mRNA)s after infecting smooth muscle cell with the oligonucleotide of existing sequence in the gax gene.
The biological activity of the adenovirus of analysis of encoding gax gene as follows:
The smooth muscle cell of exponential phase of growth is being had or do not having in the presence of the 125ng lipofectin reagent (1ipofectin) with the adenovirus infection (48 orifice plate) that contains the GAX protein coding gene under the control of enh-hsMact promotor.Adenovirus (various extent of dilution) and fat transfection amine at room temperature are incubated 30 minutes in the substratum of serum-free.With this mixture or only with virus contact 1 hour with cell at 37 ℃.When period of infection finishes, take out the substratum that contains virus, cell is incubated in containing the DMEM substratum of 0.5%SVF.Infect per 24 hours of back and change half substratum, continue insulation 48 hours to obtain entering the cell of S phase with growth medium.In second half culture, add weak mitogenesis substratum to keep cell in stationary phase.With the Alamar method back 72 hours of infection to various cell countings.
Table I
| Plasmid (promotor) | |||||
| Plasmid-free (-) | pCMV-leadTK (CMV-leadTK) | phSMact (hSMact) | pXL3130 (Enh-hSMact) | pXL3131 (hnE-hSMact) | |
| Rabbit SMC | ＜0.01 | 100.00 | 0.67±0.06 | 107.10±11.23 | 77.9±7.93 |
| ECV304 | ＜0.01 | 100.00 | 0.12±0.05 | 5.19±0.99 | 4.68±0.77 |
| C2C12 | ＜0.01 | 100.00 | 0.11±0.01 | 4.48±0.28 | 2.81±0.09 |
| HeLa | ＜0.01 | 100.00 | 0.03±0.01 | 10.31±1.13 | 8.00±0.19 |
| NIH3T3 | ＜0.01 | 100.00 | 0.08±0.01 | 4.25±0.08 | 3.66±0.28 |
| TU182 | ＜0.01 | 100.00 | 0.03±0.00 | 6.15±0.47 | 8.46±0.41 |
| 293 | ＜0.01 | 100.00 | 0.04±0.00 | 0.77±0.07 | 0.72±0.07 |
Table II
| Plasmid (promotor) | |||||
| Plasmid-free (-) | pCMV-leadTK (CMV-leadTK) | pmSM22 (mSM22) | pXL3152 (Enh-mSM22) | pXL3153 (hnE-mSM22) | |
| Rabbit SMC | ＜0.01 | 100.00 | 2.13±0.29 | 94.61±13.43 | 100.21±23.20 |
| ECV304 | ＜0.01 | 100.00 | 0.42±0.13 | 13.81±0.35 | 11.83±1.95 |
| C2C12 | ＜0.01 | 100.00 | 0.20±0.01 | 11.19±1.84 | 8.37±0.08 |
| HeLa | ＜0.01 | 100.00 | 0.04±0.01 | 12.87±1.04 | 15.90±1.92 |
| NIH3T3 | ＜0.01 | 100.00 | 0.32±0.01 | 9.49±0.41 | 9.59±0.53 |
| TU182 | ＜0.01 | 100.00 | 0.08±0.01 | 25.78±3.04 | 26.37±0.98 |
| 293 | ＜0.01 | 100.00 | 0.09±0.00 | 2.29±0.02 | 1.78±0.03 |
＜110〉RHONE-POULENC RORER＜120〉＜130〉PCT＜140〉＜141〉＜150〉FR9812000＜151〉1998-09-25＜160〉10＜170〉PatentIn Ver.2.1＜210〉1＜211〉23＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 23 )＜400〉1gatggtccct acttatgctg cta 23＜210〉2＜211〉23＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 23 )＜400〉2cttccatcat accaaactac ata 23＜210〉3＜211〉32＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 32 )＜400〉3ctgctaaatt gctcgaggac aaattagaca aa 32＜210〉4＜211〉33＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 33 )＜400〉4ccctgacaaa gcttggctgg gctgctccac tgg 33＜210〉5＜211〉30＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 30 )＜400〉5atcgacgcgt gcccgttaca taacttacgg 30＜210〉6＜211〉38＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 38 )＜400〉6atcgacgcgt ccgctcgagc gtcaatgggg cggagttg 38＜210〉7＜211〉36＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 36 )＜400〉7ccaggctgca ctcgagacta gttcccacca actcga 36＜210〉8＜211〉36＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 36 )＜400〉8tcgtttgaag cttggaagga gagtagcttc ggtgtc 36＜210〉9＜211〉30＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 30 )＜400〉9cccgttacat aacttacggt aaatggcccg 30＜210〉10＜211〉30＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_＜222〉 ( 1 ) .. ( 23 )＜400〉10gggacgcgct tctacaaggc gctggccgaa 30
Claims (18)
1. hybrid promoter, it comprises:
The enhancing subarea of-omnipresence type strong promoter/enhanser all or part of and
-allow promoter region specific expressed in smooth muscle cell.
2. the hybrid promoter of claim 1 is characterized in that strengthening the subarea and is selected from the enhancing subarea of cytomegalovirus immediate early gene (CMV-IE), the enhancing subarea of Rous sarcoma virus LTR (LTR-RSV), the enhancing subarea of SV40 virus and the enhancing subarea of EF1 α gene.
3. the hybrid promoter of claim 1 is characterized in that strengthening the enhancing subarea that the subarea is a cytomegalovirus immediate early gene (CMV-IE), the enhancing subarea of preferred human cytomegalic inclusion disease virus immediate early gene (hCMV-IE).
4. the hybrid promoter of claim 1, it is all or part of to it is characterized in that promoter region comprises the promotor of smooth muscle cell α-Ji Dongdanbai encoding gene (SMact) or SM22 gene.
5. hybrid promoter, it comprises:
The enhancing subarea of-human cytomegalic inclusion disease virus immediate early gene (hCMV-IE) all or part of and
The promotor of-smooth muscle cell α-Ji Dongdanbai encoding gene (SMact) is all or part of.
6. hybrid promoter, it comprises:
The enhancing subarea of-human cytomegalic inclusion disease virus immediate early gene (hCMV-IE) all or part of and
-SM22 gene promoter all or part of.
7. the hybrid promoter of claim 1 is characterized in that promoter region comprises a basic promotor and one and brings tissue-specific sequence, and described sequence is from SMact promotor and/or SM22 promotor.
8. expression cassette, it comprises the coding target RNA under the hybrid promoter control that is in one of claim 1-7 or the nucleic acid of polypeptide.
9. the expression cassette of claim 8 is characterized in that it also comprises transcription termination signal in addition.
10. claim 8 or 9 expression cassette is characterized in that described nucleic acid encoding is selected from following albumen: participate in albumen and transcription factor that the albumen of cell cycle, the albumen of cell death inducing, the albumen that can change smooth muscle cell proliferation, induction of vascular generate.
11. comprise the carrier of the expression cassette of the hybrid promoter of claim 1 or claim 8.
12. the carrier of claim 11 is characterized in that it is plasmid, clay or any not by the DNA of viral capsidization.
13. the carrier of claim 11 is characterized in that it is a recombinant virus, preferably from adenovirus, retrovirus, simplexvirus or adeno associated virus.
14. contain the carrier of claim 12 and the composition of chemistry or biochemical transfering reagent.
15. contain the recombinant virus of claim 13 and the composition of physiologically acceptable carrier.
16. the cell of transforming by the carrier of the expression cassette of claim 8 or claim 11.
17. the hybrid promoter of one of claim 1-7 is used for application in the composition of smooth muscle cell selective expression nucleic acid in preparation.
18. the hybrid promoter of one of claim 1-7 is used for expressing and the not application in the composition of express nucleic acid in the adjacent epithelial cell in blood vessel at smooth muscle cell in preparation.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9812000A FR2783839B1 (en) | 1998-09-25 | 1998-09-25 | USE OF HYBRID SPECIFIC PROMOTERS TO CONTROL TISSUE EXPRESSION |
| FR98/12000 | 1998-09-25 | ||
| US12329899P | 1999-03-04 | 1999-03-04 | |
| US60/123,298 | 1999-03-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1326506A true CN1326506A (en) | 2001-12-12 |
Family
ID=26234566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99811334A Pending CN1326506A (en) | 1998-09-25 | 1999-09-23 | Use of specific hybrid promoters for controlling tissue expression |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP1115857A1 (en) |
| JP (1) | JP2002525109A (en) |
| KR (1) | KR20010075338A (en) |
| CN (1) | CN1326506A (en) |
| AU (1) | AU775717B2 (en) |
| CA (1) | CA2343922A1 (en) |
| HU (1) | HUP0105230A3 (en) |
| IL (1) | IL142107A0 (en) |
| WO (1) | WO2000018908A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004502426A (en) | 2000-07-05 | 2004-01-29 | トランジェーヌ、ソシエテ、アノニム | Chimeric promoter controlling smooth muscle expression |
| EP1310561A1 (en) * | 2001-11-09 | 2003-05-14 | Transgene S.A. | Chimeric promoters for controlling expression in skeletal muscle cells |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5266490A (en) * | 1991-03-28 | 1993-11-30 | Regeneron Pharmaceuticals, Inc. | Mammalian expression vector |
| US5298422A (en) * | 1991-11-06 | 1994-03-29 | Baylor College Of Medicine | Myogenic vector systems |
| DK0787200T3 (en) * | 1994-10-28 | 2005-08-15 | Univ Pennsylvania | Improved adenovirus and methods for its use |
| CA2274314C (en) * | 1996-12-02 | 2007-03-13 | Genemedicine, Inc. | Insulin-like growth factor i (igf-i) expression system and methods of use |
-
1999
- 1999-09-23 HU HU0105230A patent/HUP0105230A3/en unknown
- 1999-09-23 IL IL14210799A patent/IL142107A0/en unknown
- 1999-09-23 EP EP99943036A patent/EP1115857A1/en not_active Withdrawn
- 1999-09-23 CN CN99811334A patent/CN1326506A/en active Pending
- 1999-09-23 AU AU56324/99A patent/AU775717B2/en not_active Ceased
- 1999-09-23 JP JP2000572355A patent/JP2002525109A/en not_active Withdrawn
- 1999-09-23 WO PCT/FR1999/002265 patent/WO2000018908A1/en not_active Application Discontinuation
- 1999-09-23 KR KR1020017003768A patent/KR20010075338A/en not_active Application Discontinuation
- 1999-09-23 CA CA002343922A patent/CA2343922A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU775717B2 (en) | 2004-08-12 |
| WO2000018908A1 (en) | 2000-04-06 |
| EP1115857A1 (en) | 2001-07-18 |
| JP2002525109A (en) | 2002-08-13 |
| IL142107A0 (en) | 2002-03-10 |
| HUP0105230A2 (en) | 2002-04-29 |
| AU5632499A (en) | 2000-04-17 |
| HUP0105230A3 (en) | 2003-10-28 |
| CA2343922A1 (en) | 2000-04-06 |
| KR20010075338A (en) | 2001-08-09 |
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