CN1250477A - Tissue specific promoters - Google Patents
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- CN1250477A CN1250477A CN98803248A CN98803248A CN1250477A CN 1250477 A CN1250477 A CN 1250477A CN 98803248 A CN98803248 A CN 98803248A CN 98803248 A CN98803248 A CN 98803248A CN 1250477 A CN1250477 A CN 1250477A
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Abstract
本发明提供了遗传构建体,该构建体提供了在特异的组织中编码序列的优选表达。构建体包括下面的元件:(1)来自组织特异性基因的类固醇效应性启动子,(2)编码序列和(3)SV40增强子。元件排列如下:来自组织特异性基因的类固醇效应性启动子在编码序列的上游并且SV40增强子位于编码序列的3’,或者SV40增强子在编码序列的上游并且来自组织特异性基因的类固醇效应性启动子定位于SV40增强子和编码序列之间,或者来自组织特异性基因的类固醇效应性启动子在编码序列的上游并且SV40增强子定位于编码序列的内含于内。在构建体中启动子的类固醇效应水平比其在天然状态的类固醇效应水平低。The present invention provides genetic constructs that provide for preferential expression of coding sequences in specific tissues. The construct included the following elements: (1) a steroid-responsive promoter from a tissue-specific gene, (2) a coding sequence and (3) an SV40 enhancer. Elements were arranged as follows: a steroid-responsive promoter from a tissue-specific gene upstream of the coding sequence and an SV40 enhancer 3' to the coding sequence, or an SV40 enhancer upstream of the coding sequence and a steroid-responsive promoter from a tissue-specific gene The promoter was positioned between the SV40 enhancer and the coding sequence, or the steroid-responsive promoter from the tissue-specific gene was upstream of the coding sequence and the SV40 enhancer was positioned inclusive of the coding sequence. The level of steroid effect of the promoter in the construct is lower than that of its native state.
Description
Invention field
The present invention relates to the associating of prostate gland gene promoter and general enhanser SV40 enhanser, thereby be created in and activatedly in the prostatic cell do not rely on androgenic promotor substantially, these prostatic cells they growth and existence in be do not rely on androgenic.
Background of invention
Leading many genes of expressing in male prostate gland have been identified.Utilize rotaring dyeing technology or in transgenic mice after the expressing gene after deliberation the promotor and the regulatory region of many such genes.The gene that great majority are accredited as prostate specific is that male sex hormone is derivable, and the aspect of their function has at length been studied.So, antigen (PSA) (Cleutjens, the people such as Vaneekelen of prostate-specific, 1996, Riegman, people such as Vliestra, 1991), people's gland kallikrein (KLK2) (Murtha, people such as Tindall, 1993), rat prostate steroid binding protein matter (PSBP) (Claessens, people such as Rushmere, 1990; Rushmere, people such as Parker, 1987), Probasin (Pb) (Kasper, people such as Rennie, 1990; Rennie, people such as Bruchovsky, 1993), with prostate acid phosphatase gene (Virkkunen, people such as Hedberg, 1994), in regulatory element in the intron of P of Rats SBPC3 (1) gene, regulate the abduction delivering of having identified the male sex hormone response element in the albumen (Ho, people such as Marschke, 1993) with rat 20 kilodalton male sex hormones and/or in conjunction with the importance of androgen receptor.
Though the organizing specific factor in conjunction with the zone of the PSBP C3 gene promoter and first intron has obtained identifying, but giving the element of the prostate specific that is different from the male sex hormone responsiveness of expression, participation also do not have fine evaluation (Celis, people such as Claessens, 1993; Zhang, people such as Parker, 1990).P of Rats SBP C (3) gene with 4kb upstream and 2kb downstream side joint sequence tissue specificity ground and under suitable hormone control, obtain expressing (Allison, people such as Zhang, 1989) in transgenic mice.Is used to express SV40T-antigen and can causes tumor of prostate, but expression not height-limited system that other error state (ERST) is common (Maroulakou, people such as Anver, 1994) from the 5kb upstream of rat PSBP C3 (1) gene.Utilize the definite probasin of research of transgenic mice and the zone of PSBP C (3) gene can give prostate specific.
PSA and probasin regulatory region are that two researchs are maximum in the prostate gland expressing gene.The 430bp district that has determined the upstream of rat probasin gene can give prostate specific (Greenberg, people such as Demayo, 1994 that reporter gene is expressed; Matusik; WO9403594); When being used for the antigenic target of SV40T and expressing, (Greenberg, people such as Demayo, 1995) take place in tumor of prostate specifically.This expression is not always special, but specificity is obviously by comprising from the MAR (matrix attachment regions) of chicken lysozyme gene and improve (Greenberg, people such as Demayo, 1994).The 430bp promoter region is replied male sex hormone consumingly and is induced, and has obtained identifying (Claessens, people such as Rushmere, 1990 in conjunction with the male sex hormone response element of androgen receptor (AR); Kasper, people such as Rennie, 1994; Matusik, WO9403594; Rennie, people such as Bruchovsky, 1993).Negative regulatory region between base-426 and-286 has also obtained identifying (Rennie, people such as Bruchovsky, 1993).
The PSA upstream (to-630bp) also the strong male sex hormone responsiveness promotor of conduct works and has also identified the male sex hormone response element.But this district is not enough to instruct organizing specific expression in transgenic mice.Utilize this 630bp people PSA promoter region in transgenic mice, to express and activatory Ha-ras oncogene causes the growth (Schaffner, people such as Barrios, 1995) of sialisterium rather than tumor of prostate.In 4 to 5kb districts of transcription initiation site upstream, identified the enhancing subarea recently.Proved that the PSA enhanser can be used as male sex hormone and can induce enhanser and show tangible cell type specificity (Henderson, WO9519434 in conjunction with the PSA promotor; Schuur, people such as Henderson, 1996).People such as same Pang have reported to compare with open sequence when promoter region from the isolating grade of prostate cancer patient contains 7 sudden changes, and is overactive (Belldegrun and Pang, WO9614875 in prostate cancer cell line LNCaP; Pang, people such as Taneja, 1995).
Virus SV40 enhanser is the enhanser of first evaluation, and is identified widely.No matter in the upstream of gene or the downstream it have from various promotor and strengthen the characteristic of expressing, and function is all arranged at both direction.The SV40 enhanser has been used for the report limited amount that is connected with steroid responsiveness promotor.For example, Israel and Kaufman (Israel and Kaufman1989) have reported the expression of induced by dexamethasone in the construct (reaching 17 times), and this construct is with SV40 enhanser and the glucocorticosteroid response element combination of adenovirus major late promoter with the different copy numbers that originate from mouse mastocarcinoma tumour virus LTR promotor/enhanser.People such as Wynshaw-Boris (Wynshaw-Boris, people such as Short, 1986) have found that the SV40 enhanser is less to the influence of the basic transcription level (about 6 times) of the glucocorticoid inducible of rat phosphoenolpyruvate carboxykinase gene.Find sequence mediation in the gene of rat growth hormone gene 3 to 5 times of glucocorticosteroids to induce when existing or not having the SV40 enhanser be suitable (Birnbaum and Baxter1986).In another case, the glucocorticosteroid effect district that observes the tyrosine aminotransferase gene can give 2 to 3 times of glucocorticoid inducibles of SV40 enhanser/early promoter (Grange, people such as Roux, 1989).These examples show when SV40 enhanser association class sterol responsiveness promotor exists, have seen successive steroid effect usually.
Male sex hormone inductive gene probasin that the inventor has expressed SV40 enhanser and two prostate glands and the promoter region of PSA are combined in the various constructs.The substance that they have observed the inductive basic horizontal of promotor increases, and corresponding male sex hormone induced activity level does not increase.So promotor/enhanser associating now is not that male sex hormone relies on substantially, but has kept their specificity mode for the expression in prostate gland and the non-prostatic cell.So the probasin/SV40 enhanser is united and has been shown substantial prostate specific, and the PSA/SV40 enhanser to unite be random.
Brief summary of the invention
Therefore, comprise the genetic constructs that encoding sequence preferential expression in specific tissue is provided in a first aspect of the present invention, this construct comprises the steroid responsiveness promotor of following element (1) from tissue-specific gene, (2) encoding sequence, (3) SV40 enhanser, element arrangements is as follows:
Steroid responsiveness promotor from tissue-specific gene is positioned at 3 ' of encoding sequence at the upstream and the SV40 enhanser of encoding sequence, perhaps
The SV40 enhanser is positioned between SV40 enhanser and the encoding sequence, perhaps in the upstream of encoding sequence and from the steroid responsiveness promotor of tissue-specific gene
Steroid responsiveness promotor from tissue-specific gene is positioned in the intron of encoding sequence at the upstream and the SV40 enhanser of encoding sequence; Wherein promotor native state than it in construct has lower steroid effect level.
Aspect second of the present invention, comprised the steroid responsiveness promotor that contains from tissue-specific gene, with the SV enhanser and the hereditary box that can insert the insertion site of encoding sequence, described insertion site in abutting connection with and be positioned at the downstream of promotor, wherein the steroid responsiveness level that has than native state in box of promotor is low.
Of the present invention this on the one hand, the SV40 enhanser can be in the upstream of organizing specific promotor or opposite.
In embodiment preferred of the present invention, be the male sex hormone responsiveness from the promotor of tissue-specific gene, equally preferably tissue-specific gene is a prostate-specific.
In other embodiment preferred, the organizing specific promotor is the probasin promotor, Pb430 particularly as described herein or Pb286.In another embodiment, promotor is the PSA promotor, PSA630 particularly as herein described.
In a third aspect of the present invention, comprise the carrier of the hereditary box of the genetic constructs that contains a first aspect of the present invention or a second aspect of the present invention.
At at present preferred carrier is adenovirus hominis 5 types or sheep adenovirus.
Treatment cancer, particularly prostate cancer are provided in another aspect of this invention, and the method for bladder cancer and mastocarcinoma comprises and utilizes construct of the present invention to carry out gene therapy.The utilization of these constructs can be in gene therapy combines the treatment of male sex hormone surgical blanking and is used to not rely on male sex hormone or depends on androgenic prostate cancer with the expression of therapeutic gene.
By this specification sheets, unless need in the literary composition that explanation is arranged in addition, vocabulary " contains " and is interpreted as referring to contain described key element, or the integration of key element or set or do not get rid of the integration of any other key element or the integration or the set of key element or integration.
Detailed description of the invention
In order more to be expressly understood characteristic of the present invention, its preferred form is described with reference to following unrestricted embodiment.
Description of drawings
Figure 1A has shown the linear graph of plasmid pCATSAT.Shown restriction enzyme sites H (HindIII), Sp (SphI), P (PstI), S (SalI), X (XbaI), and BamHI; Amp and ori are respectively plasmid ampicillin resistance gene and replication origin.E.C. 2.3.1.28 (CAT) and serine acetyltransferase gene and rous sarcoma virus promoter (RSV) and from the polyadenylation district of human growth hormone gene or SV40.
Figure 1B has shown a plurality of promotors/enhanser construct of making in the pCATSAT carrier.The upstream promoter district of CAT gene is from probasin (Pb430 or Pb286) or PSA (Psa630) gene.The SV40 enhanser is expressed as black box, and its direction indication is the direction in NcoI (N) site.
Fig. 2 has shown the sequence of SV40 enhanser.BstYI site that is used to clone and single NcoI site underscoring.
Fig. 3 has shown the transcriptional activity of construct in a plurality of cell types that contains the Pb430 promotor.
Fig. 4 has shown the transcriptional activity of construct in a plurality of cell types that contains 286 base pair probasin promotors.
Fig. 5 has shown the transcriptional activity of construct in a plurality of cell types that contains 630 base pair PSA promotors.
Fig. 6 has shown recombinant adenovirus Ad5SVbPb430PNP.In the Ela/6 district of the disappearance of adenovirus hominis 5 types, insert the box that contains SVbPb430 enhancers/promoters, intestinal bacteria purine nucleoside phosphatase gene (PNP) and SV40 polyadenylic acid district (SVpolyA).Shown the box of equal value that contains PSA630 base pair promotor that is included among the virus of A d5PSAPNP in the following bracket.
Fig. 7 A has shown and has utilized Ad5SVbPb430PNP (SVPb) or Ad5PSAPNP and the treatment of the 6MPDR bonded influence for the PC3 cell viability.Viability is measured as the metabolic activity with respect to the contrast that does not have virus or medicine, the MOI-infection multiplicity.
Fig. 7 B has shown that virus adds the influence of medicine processing to the MRC5 cell.
Fig. 7 C has shown that virus adds the influence of medicine processing to the HepG2 cell.
Embodiment
The plasmid construction body
In Fig. 1 graphic representation strengthen the following plasmid in subarea with different positions and direction in conjunction with the promoter region and the SV40 of rat probasin and people PSA gene.Utilize basic plasmid pCATSAT to prepare all plasmids.This plasmid is by inserting with reference to gene in the BamHI site, and serine acetyltransferase (SAT) is derived from the basic plasmid of pCAT (Promega).This is under the control of the rous sarcoma virus promoter of generally expressing (RSV) with reference to reporter gene and contains 3 ' polyadenylic acid district from human growth hormone gene.As shown in the figure between SV40 and human growth hormone 3 ' polyadenylic acid district regeneration one BamHI site.
Enter pCATSAT to produce Pb430 and Pb286 construct respectively by PCR from Probasin sequence and the clone that rat genomic dna separates from the SacI site of-426 (HindIII) or-286 to+28.For Pb430, utilize in+28 SacI site and the site of mixing 5 ' primer, the PCR fragment is gone into the SacI site of pBluescriptSK+ as the SacI fragment cloning.Clone again as the HindIII-XbaI fragment then and enter pCATSAT.Utilize the site that is included in respectively in 5 ' primer and the 3 ' primer, the Pb286PCR fragment is gone into pBluescriptSK+ as EcoRI to the SpeI fragment cloning; Then, enter the pCATSAT of HindIII-XbaI cutting as the HindIII-SpeI fragment cloning.The PSA630 plasmid contains the sequence from EcoRI site (630) to HindIII site (+6) that is positioned at PSA transcription initiation site 5 ', and this sequence is isolating from the human gene group DNA by pcr amplification.
The SV40 enhancer sequence of separating the BstYI pieces from plasmid pCAT enhanser (Promega).Fig. 2 has shown the sequence that strengthens the subarea.Unique N coI site underscoring.The BamHI site that this fragment subclone is entered plasmid Pb430CATSAT and PSA630CATSAT is to produce plasmid Pb430CATSATSVa and Pb430CATSATSVb and PSA630CATSATSVa and PSA630CATSATSVb respectively.As shown in Figure 1, the difference of a of enhanser and b direction is the position in NcoI site.For enhanser being placed PB430 promotor upstream, BstYI enhanser fragment cloning is entered the pBluescriptSK+ (Stratagene) of BamHI cutting, clone again with the XbaI-KpnI pieces then and enter pUC19.Then, cut out, and clone the HindIII site that enters Pb430CATSAT, obtain plasmid SVaPb430CATSAT and SVbPb430CATSAT with the HindIII pieces.The BamHI site of 5 ' PCR primer is mixed in utilization, the SV40 enhanser is cloned in the front of the Pb286 promotor in the pBluescriptSK+ plasmid.Then, as shown in Figure 1, with the HindIII-SpeI pieces two enhanser directions of Pb286 front are cloned again and to enter HindIII-XbaI cracked pCATSAT and obtained plasmid SVaPb286CATSAT and SVbPb286CATSAT.
Utilize the Pb430CATSAT construct and contain the various cell types of plasmid transfection of any direction (downstream of CAT gene or be right after the upstream of probasin promotor) SV40 enhanser.
Cell type
The clone of the tumor of prostate origin of utilizing is LNCaP (obtaining from doctor L.Chung) and PC-3 (obtaining from ATCC).The LNCaP cell is the male sex hormone sensitivity and androgen receptor that express sudden change; PC-3, DU145 and TsuPr cell be do not rely on androgenic.The tumor cell line that originates from other tissue comprises mastocarcinoma cell line mcf-7 (obtaining from ATTC), hepatic cell line HepG2 cell (from doctor G.Schreiber) and bladder cancer cell lines BL13 (Russell, people such as Wass, 1989).Obtain people's 293 embryonic kidney cells that adenovirus transforms from doctor F.Graham.Obtain normal lung fibroblast MRC5 and Chinese hamster ovary line CHO K1 from doctor R.Holliday.In T substratum (Thalmann, people such as Sikes, 1996), keep the LNCaP cell.Containing 2 mmoles/rise L-glutamic acid, cultivate PC-3 among the RPMI1640 of non-essential amino acid and 10% foetal calf serum (FBS), DU145 and TsuPr MCF7 and BL13 cell.Containing 2 mmoles/rise L-glutamic acid, HepG2 and MRC5 cell have been kept among Dulbecco ' the s MEM of 0.45% glucose and 10%FBS.In the MEM that contains non-essential amino acid and 10%FBS, keep CHO and 293 cells.
Transfection
For transfection, in 35 millimeters culture dish sowing cell to 30 to 50% be paved with and utilize DOTAP (Boehringer) according to the scheme of manufacturers transfection in second day.Optimize the amount of DNA and DOTAP for each cell type.Usually, 2.5 micrograms of DNA and 15 microlitre DOTAP are mixed for culture dish of transfection.The contrast transfection contain as negative control plasmid (pBR322) DNA or as the RSVCAT of positive control and the standard mixture of RSVSAT plasmid.Explanation according to manufacturers utilizes Lipofectamine (Gibco-BRL) to carry out the MRC5 cell transfecting.The similar scheme of utilizing another cationic lipid transfection reagent CS067 and T.Lockett to provide is carried out the transfection (also can use other transfection reagent such as Lipofectamine) of DU145 and TsuPr cell.
CAT and SAT test:
After transfection, utilized PBS rinsing cell 3 times in 44-48 hour, and by blowing into 1.5 milliliters of centrifuge tubes and centrifugal collecting cell in short-term.Containing 60 microlitres, the 0.1 mol Tris HCl of 500 micro-molar concentration Pefabloc proteinase inhibitor (Boehringer), re-suspended cell precipitation among the pH7.5.By three circulating frozens and the dissolved cell that thaws, shard is 5 minutes in little whizzer.Get supernatant liquor and carry out the SAT test, heated remaining extracts 10 minutes at 65 ℃.Shard is used for CAT test (Sleigh 1986) with supernatant liquor once more.The non-heat extraction substrate that adds is used for the active test of serine acetyltransferase.Reaction mixture (20 microlitre) contains 2 or 4 microlitre cell extracts, 1 mmole/rise acetyl-CoA and contain the 200 micromoles per liter Serines (Amersham, 50 millicurie/milliliters, 150 millicuries/mmole) of 0.1 microcurie 14C Serine.Between 20 and 120 minutes, get aliquots containig, by heating 3 minutes stopped reaction at 95 ℃.By the thin-layer chromatography reaction product isolated, and carry out phosphorus imaging analysis (molecular dynamics).Utilize ImageQuant software to determine the transforming degree of Serine to acetylserine.The ratio of RSVCAT and RSVSAT can be used for proofreading and correct the promoter activity with respect to the RSV promotor in the contrast transfection of each experiment.
Promoter activity
Fig. 2 has shown the activity of the construct of the SV40 enhanser that contains Pb430 promotor and different arrangements in the different cell types.Promotor has shown low activity separately in the LNCaP prostate cell line, and single promotor has shown negligible activity in the PC-3 clone that lacks androgen receptor.The androgen receptor cotransfection causes intense stimulus to be expressed in the PC-3 cell, has shown the critical androgen-dependent of promotor.In non-prostate cell line, in mastocarcinoma cell line mcf-7 and hepatic cell line HepG2, seen slight activity.For all four constructs that contain enhanser, in most cell types, seen the enhanced expression.At PC-3, seen the most tangible enhancing in DU145 and the TsuPr prostate cell line, the expression in these clones is the 10-60% of RSV promotor.When lacking the cotransfection of androgenic acceptor, seen high-caliber expression.Really, when the androgen receptor cotransfection, find that usually perhaps activity is low, reflected the competition between binding factor or stimulation approach in the PC-3 cell.In DU145 and TsuPr clone, seen same effect (data not shown).Pb430SVa, SVaPb430 and SVbPb430 construct have shown in prostate gland PC-3 and DU145 cell than high 3.5 to the 6 times expression of any other test cell type.Clone in secretion property epidermis source has been seen the highest expression in the non-prostatic cell type among mastocarcinoma cell line mcf-7 and the bladder cancer cell lines BL13.Should be noted that in MCF-7 clone and expressed androgen receptor.For example for the SVbPb430 construct, in other clone, 293 (kidneys), low 20 times at least of the expression among MRC5 (lung fibroblast) and the HepG2 (liver).Other experiment has been presented at that the AR gene co-transfection has also activated the Pb430 promotor in most of non-prostate cell lines.With the functional similarity in the PC-3 cell, not the having or reduce a lot of demands of Pb430/SV40 enhanser to the AR gene co-transfection in conjunction with showing.So probasin promotor/SV40 enhanser is in conjunction with the basic specificity of expressing in the not dependent prostatic cell of PC-3 male sex hormone with respect to other cell type is provided.Comparing high level expression with single probasin promotor does not need to exist androgen receptor that practicality is obviously improved, because these combinations will activate in androgen independent cancer cells, and can use in the male sex hormone surgical blanking treatment that continues to design for the tumour cell of removing the male sex hormone dependence.
Utilize the Pb286CATSAT construct and at the various cell type of the plasmid transfection that contains the SV40 enhanser in the upstream of Pb286 promotor of both direction.
Fig. 4 has shown the result of transfection.As the Pb430 promotor, the Pb286 promotor is expressed in the PC3 cell very weakly, but expresses very high when utilizing the androgen receptor cotransfection.Equally, when lacking androgen receptor, Pb286 promotor and SV40 enhanser be combined in high expression level in the PC-3 cell.For SVb enhanser direction, expression be when having androgen receptor observed expression 70%, and for the SVa direction, be expressed in and reduce (being similar to the Pb430 construct) when having AR.Although express height in the non-prostatic cell type of expression ratio in the PC-3 cell, the specificity of expressing is lower than observed Pb430 construct expression specificity.So, need male sex hormone to induce this point in removal from promoter expression, the influence of enhanser is same as observed Pb430 construct.
Reduce from the expression of the PSA promoter construct that mixes the SV40 enhanser demand AR
By separately or in conjunction with the AR gene transfection, in many clones, detected from promoter construct PSA630 the expression of PSA630SVa and PSA630SVb.In Fig. 5, shown the result.In all cells type of research, when having AR, find obviously to strengthen from the expression of PSA630 promotor.But induce level difference very big.In similar all cells type, when having AR, the construct that contains the SV enhanser of 3 ' the both direction that is positioned at the CAT gene has shown that the enhancement of expressing reduces.The scope of the influence of AR in different situations is to be induced to twice from three times to suppress.Unique exception is in the MGF-7 cell, wherein strengthens respectively for SVa and SVb construct AR and expresses 5.8 and 4.3 times, and be 10 times when lacking enhanser.Although these constructs based on PSA630 have shown very low prostate specific, the SV40 enhanser is similar to influence to the Pb promotor to the influence from this male sex hormone responsiveness promoter expression, expresses promptly that to become be that male sex hormone is ind basically.
Embodiment 4
Cut out SVbPb430 promotor box with the SalI-XbaI fragment from pSVbPb430CATSAT, and flat endization.It is cloned in plasmid pXCX3/PSAPNPase (people such as Lockett again, the front of intestinal bacteria purine nucleoside phosphatase (PNP) gene 1997), this plasmid is cut with KpnI and HindIII enzyme, and flat endization is removed the PSA promotor and utilized the SpeI enzyme to cut the peace endization subsequently with being connected again.Be used to make up the recombinant human 5 type adenovirus (Fig. 6) that are carried at the PNP gene under the control of SVbPb430 adjusting sequence by reorganization then.As described in people such as Lockett (1997), carry out genetic manipulation and virus formulation.PNP gene transformation prodrug 6-methyl purine-2-deoxyribosyl nucleosides becomes toxic product 6-methyl purine.The 6-methyl purine can be by mixing cause death cell and because it is uncharged, it can spread and deadly cell (" onlooker " effect) of not expressing enzyme of RNA and DNA between cell.
Activity and the specificity of virus of A d5SVbPb430PNP and virus of A d5PSAPNP are suitable in mediation prodrug dependent cell causes death, and afterwards the PNP expression of gene is under the control of the promotor of prostate specific antigen (PSA) gene in the virus.(24 hole flat board) every hole sowing cell about 10 in 16 millimeters holes
5, after 24 hours, with the proportional range adding virus (referring to Fig. 7) of 1 to 200 plaque forming unit (pfu)/cell.Exist in independent virus, virus adds 50 micromoles per liter 6-MPDR, or 6-MPDR incubation cell when independent.Utilize AlamarBlue test after 6 days, to determine cell growth and viability by measuring metabolic activity.Do not observe the independent effect of medicine.When high dosage viral, see some effects of independent virus, and in Fig. 7 the cell-lethal effect calibration viral individual effect.
Can see that Ad5SVbPb430PNP is more effective than Ad5PSAPNP when deadly prostate gland PC-3 cell; When importing, low at least 10 times of viruses seen inhibition of equal value.Do not see same difference in the lethal effect for non-prostatic cell MRC-5 and HepG2; Two kinds of virus shown equate to the HepG2 cell inhibiting; And only see at the metabolic activity that utilizes Ad5SVbPb430PNP to infect back MRC-5 cell at the highest viral testing level (200pfu) and significantly reducing.The virus input that suitable cell-lethal effect degree needs is higher 5 to 10 times than PC-3 prostate cell line for non-prostatic cell.
So in the process of the enzyme-prodrug therapy that virus is sent, the SVbPb430 promotor provides with the PSA promotor and compared the activity that improves in prostatic cell, and has shown the obvious selectivity for prostatic cell.
Be understood that in these embodiments, these promotors/enhanser are in conjunction with being new and having higher practicality than original promotor because they can male sex hormone responsiveness and male sex hormone not the dependency prostatic cell and when lacking male sex hormone (in the cancer patient that is carrying out the treatment of male sex hormone surgical blanking) to be specific to the expression of prostatic mode controlling gene.This is particularly important in the expression of therapeutic gene in the androgen independent prostate cancer of gene therapy for example.
Be understood that construct of the present invention also can be used for surgical intervention (cutting) or the use male sex hormone is synthetic or the patient of the inhibitor of effect.Especially, such male sex hormone blocking treatment can continue and not influence the activity of these promotors basically.
What one skilled in the art should appreciate that is can produce many variations and/or modification to the present invention shown in special embodiment and do not break away from the spirit or scope of the present invention.So it is illustrative and not restrictive that the present embodiment should be considered to.
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Claims (22)
1. make encoding sequence preferential genetic constructs of expressing in specific tissue, this construct comprises following element: (1) from the steroid responsiveness promotor of tissue-specific gene, (2) encoding sequence and (3) SV40 enhanser, and element arrangements is as follows:
Steroid responsiveness promotor from tissue-specific gene is positioned at 3 ' of encoding sequence at the upstream and the SV40 enhanser of encoding sequence, perhaps
The SV40 enhanser is positioned between SV40 enhanser and the encoding sequence, perhaps in the upstream of encoding sequence and from the steroid responsiveness promotor of tissue-specific gene
Steroid responsiveness promotor from tissue-specific gene is positioned in the intron of encoding sequence at the upstream and the SV40 enhanser of encoding sequence; Wherein promotor native state than it in construct has lower steroid effect level.
2. according to the genetic constructs of claim 1, wherein from the steroid responsiveness promotor of tissue-specific gene in the upstream of encoding sequence, the SV40 enhanser is at 3 ' of encoding sequence.
3. according to the genetic constructs of claim 1, wherein the SV40 enhanser is in the upstream of encoding sequence, is positioned between SV40 enhanser and the encoding sequence from the steroid responsiveness promotor of tissue-specific gene.
4. according to the genetic constructs of claim 1, wherein from the steroid responsiveness promotor of tissue-specific gene in the upstream of encoding sequence, the SV40 enhanser is positioned in the intron in the encoding sequence.
5. according to each genetic constructs of claim 1 to 4, wherein the steroid responsiveness promotor from tissue-specific gene is the male sex hormone responsiveness.
6. according to each genetic constructs of claim 1 to 5, wherein tissue-specific gene is a prostate specific.
7. according to each genetic constructs of claim 1 to 6, wherein the steroid responsiveness promotor from tissue-specific gene is the probasin promotor.
8. according to the genetic constructs of claim 7, wherein promotor is Pb430 or Pb286.
9. according to each genetic constructs of claim 1 to 6, wherein the steroid responsiveness promotor from tissue-specific gene is the PSA promotor.
10. according to the genetic constructs of claim 9, wherein promotor is PSA630.
11. contain from the steroid responsiveness promotor of tissue-specific gene and the hereditary box in SV40 enhanser and insertion site, encoding sequence can be inserted in wherein said insertion site, insert the site in abutting connection with and be positioned at the downstream of promotor, wherein the level of the steroid responsiveness of promotor in box is lower than its native state.
12. according to the hereditary box of claim 11, wherein the SV40 enhanser is positioned the upstream of the steroid responsiveness promotor of tissue-specific gene.
13. according to the hereditary box of claim 11, wherein the SV40 enhanser is the downstream in the steroid responsiveness promotor of tissue-specific gene.
14. according to each hereditary box of claim 11 to 13, wherein the steroid responsiveness promotor of tissue-specific gene is the male sex hormone responsiveness.
15. according to each hereditary box of claim 11 to 14, wherein tissue-specific gene is a prostate specific.
16. according to each hereditary box of claim 11 to 15, wherein the steroid responsiveness promotor of tissue-specific gene is the probasin promotor.
17. according to the hereditary box of claim 16, wherein promotor is Pb430 or Pb286.
18. according to each hereditary box of claim 11 to 15, wherein the steroid responsiveness promotor of tissue-specific gene is the PSA promotor.
19. according to the hereditary box of claim 18, wherein promotor is PSA630.
20. comprise each genetic constructs or each the carrier of hereditary box of claim 11 to 19 of claim 1 to 10.
21. according to the carrier of claim 20, wherein carrier is adenovirus hominis 5 types.
22. according to the carrier of claim 20, wherein carrier is the sheep adenovirus.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPO4923A AUPO492397A0 (en) | 1997-02-03 | 1997-02-03 | Prostate specific promoters |
| AUPO4923 | 1997-02-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1250477A true CN1250477A (en) | 2000-04-12 |
Family
ID=3799227
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98803248A Pending CN1250477A (en) | 1997-02-03 | 1998-02-03 | Tissue specific promoters |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1009820A1 (en) |
| CN (1) | CN1250477A (en) |
| AU (1) | AUPO492397A0 (en) |
| CA (1) | CA2279552A1 (en) |
| WO (1) | WO1998033903A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618536A (en) * | 2008-02-14 | 2012-08-01 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence for gene therapy |
| CN102628041A (en) * | 2008-02-14 | 2012-08-08 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence used for gene therapy |
| US8569051B2 (en) | 2008-02-14 | 2013-10-29 | Mogam Biotechnology Research Institute | Expression vector for gene therapy |
| CN102634515B (en) * | 2008-02-14 | 2013-11-06 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence used for gene therapy |
| CN109988759A (en) * | 2017-12-29 | 2019-07-09 | 上海细胞治疗研究院 | A kind of chimeric promoters in T cell with high transcriptional activity |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU773906B2 (en) | 1999-03-01 | 2004-06-10 | Commonwealth Scientific And Industrial Research Organisation | Regulatory constructs comprising intron 3 of prostate specific membrane antigen gene |
| ES2385492T3 (en) | 2004-04-02 | 2012-07-25 | Board Of Regents, The University Of Texas System | Specific cancer promoters |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919652A (en) * | 1994-11-09 | 1999-07-06 | The Regents Of The University Of California | Nucleic acid molecules comprising the prostate specific antigen (PSA) promoter and uses thereof |
-
1997
- 1997-02-03 AU AUPO4923A patent/AUPO492397A0/en not_active Abandoned
-
1998
- 1998-02-03 CA CA002279552A patent/CA2279552A1/en not_active Abandoned
- 1998-02-03 CN CN98803248A patent/CN1250477A/en active Pending
- 1998-02-03 WO PCT/AU1998/000057 patent/WO1998033903A1/en not_active Application Discontinuation
- 1998-02-03 EP EP98901256A patent/EP1009820A1/en not_active Withdrawn
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618536A (en) * | 2008-02-14 | 2012-08-01 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence for gene therapy |
| CN102628041A (en) * | 2008-02-14 | 2012-08-08 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence used for gene therapy |
| CN102628041B (en) * | 2008-02-14 | 2013-09-11 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence used for gene therapy |
| CN102618536B (en) * | 2008-02-14 | 2013-10-16 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence for gene therapy |
| US8569051B2 (en) | 2008-02-14 | 2013-10-29 | Mogam Biotechnology Research Institute | Expression vector for gene therapy |
| CN102634515B (en) * | 2008-02-14 | 2013-11-06 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence used for gene therapy |
| US8617875B2 (en) | 2008-02-14 | 2013-12-31 | Mogam Biotechnology Research Institute | Expression vector suitable for expression of a coding sequence for gene therapy |
| US8628956B2 (en) | 2008-02-14 | 2014-01-14 | Mogam Biotechnology Research Institute | Expression vector suitable for expression of a coding sequence for gene therapy |
| CN109988759A (en) * | 2017-12-29 | 2019-07-09 | 上海细胞治疗研究院 | A kind of chimeric promoters in T cell with high transcriptional activity |
Also Published As
| Publication number | Publication date |
|---|---|
| AUPO492397A0 (en) | 1997-02-27 |
| WO1998033903A1 (en) | 1998-08-06 |
| CA2279552A1 (en) | 1998-08-06 |
| EP1009820A1 (en) | 2000-06-21 |
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