TISSUE SPECIFIC PROMOTERS

Abstract: The present invention provides a genetic construct providing preferential expression in specific tissue of a coding sequence. The construct includes the following elements (1): a steroid responsive promoter from a tissue specific gene, (2) a coding sequence, and (3) an SV40 enhancer. The elements are arranged as follows: the steroid responsive promoter from the tissue specific gene being upstream of the coding sequence and the SV40 enhancer being 3' of the coding sequence, or the SV40 enhancer being upstream of the coding sequence and the steroid responsive promoter from the tissue specific gene being positioned between the SV40 enhancer and the coding sequence, or the steroid responsive promoter from the tissue specific gene being upstream of the coding sequence and the SV40 enhancer being positioned within an intron within the coding sequence. The promoter has a lowered level of steroid responsiveness in the construct than in its native state.

Description: Tissue Specific Promoters FIELD OF THE INVENTION The present invention is concerned with combinations of prostate gene promoters with a general enhancer, the SV40 enhancer, giving rise to essentially androgen-independent promoters active in prostate cells which are androgen-independent in their growth and survival. BACKGROUND OF THE INVENTION A number of genes have been identified which are expressed predominantly in the prostate in males. Promoters and regulatory regions of a number of such genes have been stvidied, either ising transfection techniques or by following gene expression in transgenic mice. Most of the genes identified as prostate-specific are androgen-inducible and this aspect of their function has been studied in some detail. Thus the importance of androgen response elements for induced expression and/or binding of androgen receptor have been characterised in the prostate specific antigen (PSA) (Cleutjens, Vaneekelen et al. 1996; Riegman, Vliestra et al. 1991), human glandular kallikrein (KLK2) (Murtha, Tindall et al. 1993), rat prostatic steroid binding proteins (PSBP) (Claessens, Rushmere et al. 1990; Rushmere, Parker et al. 1987), probasin (Pb) (Kasper, Rennie et al. 1994; Rennie, Bruchovsky et al. 1993) and prostatic acid phosphatase genes (Virkkunen, Hedberg et al. 1994) and in regulatory elements in the introns of the rat PSBP C3(l) gene (Celis, Claessens et al. 1993) and the rat 20-KDa androgen regulated protein (Ho, Marschke et al. 1993). Elements involved in conferring prostate-specificity of expression per se, as distinct from androgen responsiveness, have not been well characterised, though tissue-specific factors binding to regions of the PSBP C3 gene promoter and 1st intron have been identified (Celis, Claessens et al. 1993; Zhang, Parker et al. 1990). The gene for rat PSBP C(3) with 4 kb upstream and 2 kb downstream flanking sequences is expressed tissue- specifically and with appropriate hormonal control in transgenic mice (Allison, Zhang et al. 1989). The use of a 5kb upstream region from the rat PSBP C3(l) gene to express the SV40 T-antigen could elicit prostate tumours, but expression was not highly restricted and other abnormalities were common (Maroulakou, Anver et al. 1994). Studies with transgenic mice have established that regions of the probasin and PSBP C(3) genes can confer prostate specificity. The PSA and probasin regulatory regions are the two most studied among prostate-expressed genes. It has been established that a 430 bp region upstream of the rat probasin gene is able to confer prostate specificity of expression on a reporter gene (Greenberg, Demayo et al. 1994; Matusik; WO9403594); when used to target expression of the SV40 T-antigen, prostate tumours develop specifically (Greenberg, Demayo et al. 1995). This expression is not totally specific but specificity is significantly improved by the inclusion of MAR (matrix attachment regions) from the chick lysozyme gene (Greenberg, Demayo et al. 1994). The 430 bp promoter region is strongly responsive to androgen induction and androgen response elements which bind the androgen receptor (AR) have been characterised (Claessens, Rushmere et al. 1990; Kasper, Rennie et al. 1994; Matusik, WO9403594; Rennie, Bruchovsky et al. 1993). A negative regulatory region between bases -426 and -286 has also been identified (Rennie, Bruchovsky et al. 1993). The PSA upstream region (to -630 bp) also acts as a strongly androgen responsive promoter and androgen response elements have also been characterised. This region is not sufficient to direct tissue specific expression in transgenic mice, however. Use of the 630 bp human PSA promoter region to express an activated Ha-ras oncogene in transgenic mice led to the development of salivary gland and not prostate tumours (Schaffner, Barrios et al. 1995). An enhancer region has recently been identified in the region 4 to 5kb upstream of the transcription start site. The PSA enhancer has been shown to act as an androgen-inducible enhancer and in combination with the PSA promoter to display significant cell-type specificity (Henderson, W09519434; Schuur, Henderson et al. 1996). Also Pang et al. have reported that the equivalent promoter region isolated from a prostate cancer patient contained 7 mutations compared to the published sequence and was highly active in the prostate cancer cell line LNCaP (Belldegrun and Pang, W09614875; Pang, Taneja et al. 1995). The viral SV40 enhancer was the first enhancer identified and it has been extensively characterised. It has the properties of enhancing expression from a variety of promoters whether it is upstream or downstream of the gene and is functional in either orientation. There are a limited number of reports where the SV40 enhancer has been used in conjunction with a steroid-responsive promoter. For example Israel and Kaufman (Israel and Kaufman 1989) reported substantial dexamethasone induction of expression (up to 17 fold) in constructs which combined the SV40 enhancer and adenovirus major late promoter with different copy numbers of glucocorticoid response elements derived from the mouse mammary tumour virus LTR promoter/enhancer. Wynshaw-Boris et al. (Wynshaw-Boris, Short et al. 1986) found that the SV40 enhancer had little effect on basal level transcription or glucocorticoid-inducibility (about 6- fold) of the rat phosphoenolpyruvate carboxykinase gene. The 3 to 5-fold glucocorticoid indLiction mediated by intragenic seq iences of the rat growth hormone gene was found to be equivalent in the presence or absence of the SV40 enhancer (Birnbaum and Baxter 1986). In another case it was observed that the glucocorticoid response region of the tyrosine amino transferase gene was able to confer 2 to 3 fold glucocorticoid ind icibility on the SV40 enhancer/early promoter (Grange, RO X et al. 1989). These examples indicate that continued steroid responsiveness is normally seen when the SV40 enhancer is present in conjunction with a steroid-responsive promoter. The present inventors have combined, in various constructs, the viral SV40 enhancer with the promoter regions of two prostate-expressed, androgen-induced genes -probasin and PSA. They have observed a substantial increase in the basal level of induction of the promoters without a corresponding increase in the androgen-induced level of activity. The promoter/enhancer combinations are thus now essentially androgen-independent, but maintain their specificity profile with respect to expression in prostate and non-prostate cells. Thus the probasin/SV40 enhancer combinations show svibstantial prostate specificity, while PSA/SV40 enhancer combinations are more promiscuous. SUMMARY OF THE INVENTION Accordingly in a first aspect the present invention consists in a genetic construct providing preferential expression in specific tissue of a coding seqvience, the construct including the following elements (1) a steroid responsive promoter from a tissue specific gene, (2) a coding sequence, and (3) an SV40 enhancer, the elements being arranged as follows: the steroid responsive promoter from the tissLie specific gene being vipstream of the coding sequence and the SV40 enhancer being 3' of the coding sequence, or the SV40 enhancer being upstream of the coding sequence and the steroid responsive promoter from the tissue specific gene being positioned between the SV40 enhancer and the coding seqLience, or the steroid responsive promoter from the tissue specific gene being upstream of the coding sequence and the SV40 enhancer being positioned within an intron within the coding sequence; wherein the promoter has a lowered level of steroid responsiveness in the construct than in its native state. In a second aspect the present invention consists in a genetic cassette comprising a steroid responsive promoter from a tissLie specific gene and an SV40 enhancer and an insertion site into which a coding sequence can be inserted, the insertion site being adjacent to and downstream of the promoter, wherein the promoter has a lowered level of steroid responsiveness in the cassette than in its native state. In this aspect of the invention the SV40 enhancer may be upstream of the tissue specific promoter or vice versa. In a preferred embodiment of the present invention the promoter from the tissue specific gene is androgen-responsive. It is also preferred that the tissue specific gene is prostate specific. In further preferred embodiments the tissue specific promoter is the probasin promoter, in particular Pb430 or Pb286 as described herein. In another embodiment the promoter is the PSA promoter, in particular PSA630 as described herein. In a third aspect the present invention consists in a vector including the genetic construct of the first aspect of the present invention or the genetic cassette of the second aspect of the present invention. It is presently preferred that the vector is human adenovirus Type 5 or ovine adenovirus. In a further aspect the present invention provides methods of treatment of cancers, in particular, prostate, bladder and breast, involving gene therapy using the constructs of the present invention. The use of these constracts enables the use of androgen-ablation treatment in combination with the expression of therapeutic genes in gene therapy for androgen-independent or androgen-dependent prostate cancer. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or groLip of elements or integers. DETAILED DESCRIPTION OF THE INVENTION In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following non-limiting Examples. FIGURE LEGENDS Figure 1A shows a linearised map of the plasmid pCATSAT. Restriction enzyme sites H(Hind III), Sp(Sph I), P(PstΙ), S(SalI), X(XbaΙ) and BamHI are shown; amp and ori are the plasmid ampicillin resistance gene and origin of replication respectively. The chloramphenicol acetyl transferase (CAT) and serine acetyl transferase genes are indicated as is the rous sarcoma vims promoter (RSV) and polyadenylation regions derived from the hviman growth hormone gene or SV40. Figure IB shows a niimber of promoter/enhancer constructs made in the pCATSAT vector. Promoter regions Lipstream of the CAT gene were derived from either the probasin (Pb430 or Pb286) or PSA (Psa630) genes. The SV40 enhancer is shown as a dark box, its orientation being indicated by the position of the Ncol (N) site. Figure 2 shows the sequence of SV40 enhancer. The BstYI sites used for cloning and the unique Ncol site are underlined. Figure 3 shows the transcriptional activity of constructs containing the Pb430 promoter in a number of cell types. Figure 4 shows the transcriptional activity of constmcts containing the 286 base pair probasin promoter in a number of cell types. Figure 5 shows the transcriptional activity of constracts containing the 630 base pair PSA promoter in a number of cell types. Figure 6 shows the recombinant adenoviras Ad5SVbPb430PNP. The cassette containing the SVbPb430 enhancer/promoter, the E.coli pLirine nucleoside phosphorylase gene (PNP) and SV40 polyadenylation region (SVpolyA) is inserted into the deleted Ela/6 region of the human adenovirus type 5. Below, in brackets, is shown the equivalent cassette with the PSA630 base pair promoter included in the virus AdδPSAPNP. Figure 7A shows the effect of the combination of treatment with either Ad5SVbPb430PNP (SVPb) or AdδPSAPNP in combination with 6-MPDR or PC3 cell viability. Viability was measured as metabolic activity relative to control, without virus or drag. MOI - multiplicity of infection. Figure 7B shows the effect of virus phis drag treatment on MRC5 cells. Figure 7C shows the effect of viras plus drug treatment on HepG2 cells. EXAMPLES Plasmid Constructs. The following plasmids combining the SV40 enhancer region in different positions and orientation with the promoter regions of the rat probasin and human PSA genes are diagrammed in Figure 1. All plasmids have been prepared Lising the base plasmid pCATSAT. This plasmid was derived from the plasmid pCAT-basic (Promega) by insertion into the BamHI site of a reference gene, serine acetyl transferase (SAT,) This reference reporter gene is under the control of the ubiqLiitously-expressed rous sarcoma virus promoter (RSV) and contains a 3' polyadenylation region derived from the human growth hormone gene. A BamHI site was re- generated as shown between the SV40 and Imman growth hormone 3' polyadenylation regions. Probasin sequences from -426 (Hindlll) or -286 to the Sad site at position + 28 were isolated by PCR from rat genomic DNA and cloned into pCATSAT to produce the Pb430 and Pb286 constructs, respectively. For Pb430 the PCR fragment was cloned as a Sad fragment into the Sad site of pBluescriptSK+ using the Sad site at +28 and a site incorporated into the 5' primer. It was then recloned as a Hindlll to Xbal fragment into pCATSAT. The Pb286 PCR fragment was cloned into pBluescriptSK+ as an EcoRI to Spel fragment using sites contained in the 5' and 3' primers respectively; it was then cloned as an Hindlll to Spel fragment into Hindlll-Xbal cut pCATSAT. The PSA630 plasmid contained sequences 5' of the PSA transcription start site from EcoRI site (-630) to the Hindlll site (+6) which were isolated from human genomic DNA by PCR amplification. SV40 enhancer sequences were isolated as a BstYI fragment from the plasmid pCAT-enhancer (Promega). The sequence of the enhancer region is shown in FigLire 2. The uniqLie Ncol site is underlined. This fragment was subcloned into the BamHI sites of the plasmids Pb430CATSAT and PSA630CATSAT to produce the plasmids Pb430CATSATSVa and . Pb430CATSATSVb and PSA630CATSATSVa and PSA630CATSATSVb respectively. The a and b orientations of the enhancer are distinguished by the position of the Ncol site as indicated in Figure 1. For placement of the enhancer upstream of the PB430 promoter the BstYI enhancer fragment was cloned into BamHI-cut pBluescriptSK+ (Stratagene), then re-cloned as a Xbal to Kpnl fragment into pUCl9. It was then excised as a Hindlll fragment and cloned into the Hindlll site of Pb430CATSAT to give the plasmids SVaPb430CATSAT and SVbPb430CATSAT. The SV40 enhancer was cloned in front of the Pb286 promoter in the pBluescriptSK+ plasmid using BamHI site incorporated in the 5' PCR primer. The two enhancer orientations in front of Pb286 were then re-cloned as Hindlll-Spel fragments into Hindlll- Xbal cut pCATSAT to give plasmids SVaPb286CATSAT and SVbPb286CATSAT as shown in Figure 1. Example 1: A range of cell types were transfected with the Pb430CATSAT construct and plasmids containing the SV40 enhancer in either orientation either downstream of the CAT gene or immediately upstream of the probasin promoter. Cell Types Prostate tumour-derived cell lines used were LNCaP (obtained from Dr. L. Chung) and PC-3 (obtained from the ATCC). LNCaP cells are androgen-sensitive and express a mutated androgen receptor; PC-3, DU145 and TsuPr cells are androgen independent. Tumour cell lines derived from other tissues included the breast cancer line MCF-7 (obtained from the ATTC), the liver cell line HepG2 cells (from Dr. G. Schreiber), and the bladder cancer cell line BL13 (Russell, Wass et al. 1989). Human 293 embryonic kidney cells, adenovirus transformed, were obtained from Dr. F. Graham. The normal lung fibroblast line MRC5 and the Chinese hamster ovary line CHO Kl were obtained from Dr. R. Holliday. LNCaP cells were maintained in T-medium (Thalmann, Sikes et al. 1996). PC-3, DU145 and TSLIPΓ MCF7 and BL13 cells were maintained in RPMI 1640 medium containing 2mM glutamine, non-essential amino acids and 10% fetal bovine seram (FBS). HepG2 and MRC5 cells were maintained in Dulbecco's MEM containing 2 mM glutamine, 0.45% glucose and 10% FBS. CHO and 293 cells were maintained in MEM containing non-essential amino acids and 10% FBS. Transfections For transfection, cells were seeded at 30 to 50% confluency in 35mm dishes and transfected the following day using DOTAP (Boehringer) according to the manufacturer's protocols. Amounts of DNA and DOTAP were optimised for each cell type. Generally, 2.5μg of DNA mixed with 15μl of DOTAP was used to transfect one dish. Control transfections contained either plasmid (pBR322) DNA as a negative control or a standard mix of RSVCAT and RSVSAT plasmids as a positive control. Transfection of MRC5 cells was carried out Lising Lipofectamine (Gibco-BRL) according to the manufacturer's instructions. Transfections of DU145 and TsuPr cells were done Lising another cationic lipid transfection reagent CS06 and a similar protocol provided by Dr. T. Lockett (other transfection reagents such as Lipofectamine may also be Lised). CAT and SAT assays: At 44-48hr post-transfection cells were rinsed three times with PBS, and harvested by scraping into a 1.5ml centrifuge tube and brief centrifugation. Cell pellets were resuspended in 60μl of 0.1M Tris.HCl, pH7.5, containing 500μM Pefabloc protease inhibitor (Boehringer). Cells were lysed by three cycles of freezing and thawing and debris was pelleted for 5min in a microfuge. Supernatant (20μl) was set aside for SAT assays and the remaining extract was heated at 65°C for 10 min. Debris was again pelleted and the supernatant used for CAT assays (Sleigh 1986). Non-heated extract was used for assays of serine acetyl transferase activity. Reaction mixtures (20μl) comprised 2 or 4μl of cell extract, ImM acetyl coenzyme A and 200uM serine containing O.lμCi 14C-serine (Amersham, 50 mCi/ml, 150 mCi/mmol). Aliquots were removed between 20 and 120 min and reactions were stopped by heating at 95°C for 3 min. Reactions products were separated by thin layer chromatography and subjected to phosphor image analysis (Molecular Dynamics). The extent of conversion of serine to acetylated serine was determined using ImageQuant software. The ratio of RSVCAT to RSVSAT in the control transfection in each experiment was used to normalise promoter activities relative to the RSV promoter. Prom oter Activities Activities of the constructs containing the Pb430 promoter and different arrangements of the SV40 enhancer in different cell types are shown in FigLire 2. The promoter alone shows low activity in the LNCaP prostate cell line and negligible activity in the PC-3 line which lacks an androgen receptor. Co-transfection of the androgen receptor into PC-3 cells leads to a strong stimulation of expression, indicating the critical androgen- dependence of the promoter. Among the non-prostate cell lines slight activity was seen in the MCF-7, breast, and HepG2, liver, cell lines. For all foLir enhancer-containing constructs increased expression was seen in most cell types. The most dramatic increase was seen in the PC-3 . . DU145 and TsuPr prostate cell lines where expression ranged from 10 to 60% that of the RSV promoter. This high level expression is seen in the absence of co- transfection of the androgen receptor. Indeed, when the androgen receptor was co-transfected activity was generally found to be lower in PC-3 cells, perhaps reflecting competition between binding factors or stimulatory pathways. A similar effect was seen in the DU145 and TSLIPΓ lines (data not shown). The Pb430SVa, SVaPb430 and SVbPb430 constracts showed 3.5 to 6 fold higher expression in the prostate PC-3 and DU145 cells than in any of the other cell types tested. Highest expression in non-prostate cell types was seen in the cell lines of secretory epithelial origin, the breast cancer cell line MCF-7 and the bladder cancer cell line BL13. It should be noted that the androgen receptor is expressed in the MCF-7 line. Expression in the other cell lines, 293 (kidney), MRC5 (lung fibroblast) and HepG2 (liver) was much lower, at least 20 fold, for example, for the SVbPb430 constract. Other experiments have shown that the Pb430 promoter can also be activated by AR gene co-transfection in most non-prostate cell lines. Similarly to their fLinction in PC-3 cells the Pb430/SV40 enhancer combinations show no or much reduced requirement for AR gene co-transfection. Tims the probasin promoter/SV40 enhancer combinations provide for substantial specificity for expression in the PC-3 androgen-independent prostate cells relative to other cell types. The lack of reqLiirement for the presence of androgen receptor for high level expression also provides for a significant improvement in utility compared to the probasin promoter alone, as these combinations will be active in androgen-independent cancer cells and can be used while continuing androgen ablation therapy designed to eliminate androgen- dependent tumour cells. Example 2 A range of cell types were transfected with the Pb286CATSAT constract and plasmids containing the SV40 enhancer in either orientation either immediately upstream of the Pb286 promoter. Results of transfections are shown in FigLire 4. As for the Pb430 promoter, the Pb286 promoter is very poorly expressed in PC-3 cells, but is highly expressed when co-transfected with the androgen receptor. Again, combination of the Pb286 promoter with the SV40 enhancer are highly expressed in PC-3 cells in the absence of the androgen receptor. For the SVb enhancer orientation expression was 70% of that seen in the presence of the androgen receptor, while for the SVa orientation expression was reduced in the presence of the AR (similarly to the Pb430 constracts). While expression was higher in PC-3 cells than in non-prostate cell types, the specificity of expression was less than that seen for the Pb430 constructs. The effect of the enhancer is thus similar to that seen for the Pb430 constracts in bypassing the requirement for androgen induction for expression from the promoter. Example 3. Red iced AR requirement for expression from PSA promoter constructs incorporating the SV40 enhancer. Expression from promoter constructs PSA630, PSA630SVa and PSA630SVb was examined in a number of cell lines following transfection alone or in combination with the AR gene. Results are shown in Figure 5. Expression from the PSA630 promoter was found to be significantly increased in the presence of AR in all cell types studied, though the level of indLiction varied considerably. Constructs containing the SV40 enhancer in either orientation 3' of the CAT gene showed substantially less enhancement of expression in the presence of AR in almost all cell types. The effect of AR ranged from three fold induction to two fold repression in different instances. The only exception was in MCF-7 cells where the AR enhanced expression by 5.8 and 4.3 fold for the SVa and SVb constructs respectively compared to tenfold in the absence of the enhancer. While these PSA630- based constracts show poor prostate specificity the effect of the SV40 enhancer on expression from this androgen-responsive promoter is similar to that on the Pb promoters in that expression becomes substantially androgen- independent. Example 4 The promoter cassette SVbPb430 was excised from pSVbPb430CATSAT as a Sail to Xbal fragment and blunt-ended. It was re- cloned in front of the E.coli pLirine nucleoside phosphorylase (PNP) gene in the plasmid pXCX3/PSAPNPase (Lockett et al. 1997) which had been cut with Kpnl and Hindlll, blunt-ended and re-ligated to remove the PSA promoter and subsequently cut with Spel and bkmt-ended. This was then used to construct, via recombination, a recombinant human Type 5 adenovirus carrying the PNP gene under the control of the SVbPb430 regulatory sequences (Figure 6). Genetic manipulation and virus constraction was done as described in Lockett et al. (1997). The PNP gene converts the pro-drug 6 methyl purine 2-deoxyriboside to the toxic product 6-methyl purine. 6 methyl purine can kill cells through incorporation into both RNA and DNA and because it is non-charged it can diffuse between cells and kill cells which are not expressing the enzyme (the "bystander" effect). The activity and specificity of the virus Ad5SVbPb430PNP in mediating pro-drag dependent cell killing was compared with a virus AdδPSAPNP in which the expression of the PNP gene was under the control of the promoter of the prostate specific antigen (PSA) gene. Cells were seeded at approximately 10 5 per well into 16 mm wells (24 well plates) and 24 hr later viruses were added at ratios ranging from 1 to 200 plaque forming Linits (pfu) per cell (see FigLire 7). Cells were incubated either in the presence of virus alone, virus plus 50μM 6-MPDR, or 6-MPDR alone. Cell growth and viability was determined after six days by measLirement of metabolic activity using and Alamar Blue® assay. No effect of drug alone was observed. At higher doses of virus some effect of virus alone was seen and in Figure 7 the cell killing effect is corrected for any virus alone effect. It can be seen that Ad5SVbPb430PNP is substantially more effective in killing the prostate PC-3 cells than AdδPSAPNP; an equivalent suppression was seen at viras inputs at least 10 fold lower. The same differential in killing was not seen for the non-prostate cells MRC-5 and HepG2; the two virases showed equivalent SLippression of HepG2 cells, while significant reduction in metabolic activity of MRC-5 cells following infection with Ad5SVbPb430PNP was only seen at the highest viras level tested (200 pfu). Virus inputs required for equivalent cell killing were 5 to 10 fold higher for the non-prostate cells than for the PC-3 prostate cell line. Thus, in the context of virally-delivered enzyme prodrug therapy, the SVbPb430 promoter provides for substantially improved activity in prostate cells compared with the PSA promoter, and shows significant selectivity for prostate cells. As will be appreciated from these Examples these promoter/enhancer combinations are new and have much greater utility than the original promoters in that they can be used to control expression of genes in a prostate-specific manner in both androgen-responsive and androgen- independent prostate cells and in the absence of androgens (as in cancer patients undergoing androgen-ablation treatment). This is of particular importance for example in the expression of therapeutic genes in gene therapy for androgen-independent prostate cancer. As will be Linderstood the constructs of the present invention will also be LiseiLil where patient has been treated surgically (castration) or with inhibitors of androgen synthesis or action. Specifically SLich androgen- blockade therapy can be continLied without substantially diminishing the activity of these promoters. 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Claims: Claims :- 1. A genetic construct providing preferential expression in specific tissue of a coding sequence, the constract including the following elements (1) a steroid responsive promoter from a tissue specific gene, (2) a coding sequence, and (3) an SV40 enhancer, the elements being arranged as follows: the steroid responsive promoter from the tissue specific gene being upstream of the coding sequence and the SV40 enhancer being 3' of the coding sequence, or the SV40 enhancer being Lipstream of the coding sequence and the steroid responsive promoter from the tissue specific gene being positioned between the SV40 enhancer and the coding sequence, or the steroid responsive promoter from the tissLie specific gene being Lipstream of the coding sequence and the SV40 enhancer being positioned within an intron within the coding sequence; wherein the promoter has a lowered level of steroid responsiveness in the construct than in its native state. 2. A genetic constract as claimed in claim 1 in which the steroid responsive promoter from the tissue specific gene is Lipstream of the coding seqLience and the SV40 enhancer is 3' of the coding sequence. 3. A genetic construct as claimed in claim 1 in which the SV40 enhancer is upstream of the coding seqLience and the steroid responsive promoter from the tissue specific gene is positioned between the SV40 enhancer and the coding sequence. 4. A genetic construct as claimed in claim 1 in which the steroid responsive promoter from the tissvie specific gene is Lipstream of the coding sequence and the SV40 enhancer is positioned within an intron within the coding sequence. 5. A genetic constract as claimed in any one of claims 1 to 4 in which the steroid responsive promoter from the tissue specific gene is androgen- responsive. 6. A genetic construct as claimed in any one of claims 1 to 5 in which the tissue specific gene is prostate specific. 7. A genetic construct as claimed in any one of claims 1 to 6 in which the steroid responsive promoter from the tissue specific gene is the probasin promoter. 8. A genetic constract as claimed in claim 7 in which the promoter is Pb430 or Pb286. 9. A genetic construct as claimed in any one of claims 1 to 6 in which the steroid responsive promoter from the tissue specific gene is the PSA promoter. 10. A genetic construct as claimed in claim 9 in which the promoter is PSA630. 11. A genetic cassette comprising a steroid responsive promoter from a tissLie specific gene and an SV40 enhancer and an insertion site into which a coding sequence can be inserted, the insertion site being adjacent to and downstream of the promoter, wherein the promoter has a lowered level of steroid responsiveness in the cassette than in its native state. 12. A genetic cassette as claimed in claim 11 in which the SV40 enhancer is upstream of the steroid responsive promoter of the tissLie specific gene. 13. A genetic cassette as claimed in claim 11 in which the SV40 enhancer is downstream of the steroid responsive promoter of the tissLie specific gene. 14. A genetic cassette as claimed in any one of claims 11 to 13 in which the steroid responsive promoter of the tissue specific gene is androgen- responsive. 15. A genetic cassette as claimed in any one of claims 11 to 14 in which the tissue specific gene is prostate specific. 16. A genetic cassette as claimed in any one of claims 11 to 15 in which the steroid responsive promoter of the tissue specific gene is the probasin promoter. 17. A genetic cassette as claimed in claim 16 in which the promoter is Pb430 or Pb286. 18. A genetic cassette as claimed in any one of claims 11 to 15 in which the steroid responsive promoter of the tissue specific gene is the PSA promoter. 19. A genetic cassette as claimed in claim 18 in which the promoter is PSA630. 20. A vector including the genetic construct as claimed in any one of claims 1 to 10 or the genetic cassette as claimed in any one claims 11 to 19. 21. A vector as claimed in claim 20 in which the vector is human adenovirus Type 5. 22. A vector as claimed in claim 20 in which the vector is ovine adenovirus.

Inventors: MOLLOY PETER LAURENCE, MOLLOY, PETER, LAURENCE
Applicants: COMMW SCIENT IND RES ORG, MOLLOY PETER LAURENCE, COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION, MOLLOY, PETER, LAURENCE
Application year: 1998
Main classification: C12N15
IPC codes: C12N9, C12N15