CA2266846A1 - Hex ii tumor-specific promoter and uses thereof in cancer therapy - Google Patents
Hex ii tumor-specific promoter and uses thereof in cancer therapy Download PDFInfo
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Abstract
The present invention relates to a tumor-specific promoter for use in gene targeted therapy that is differentially regulated in cancer cells, which comprises Hex II promoter. The present invention also relates to a gene construct, which include Hex II promoter in a vector selected from pCAT basic expression vector p.DELTA.ElsplB and a shuttle plasmid, and which optionally includes .beta.-gal or HSV Tk.
Description
W O 98tl3507 PCT/CA97/00691 HEX II TUMOR-~r~l~lC PRO.IJ ~ AND USES THEREOF IN
CANCER THERAPY
RACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a novel tumor-specific promoter for use in gene targeted therapy that is dif-ferentially regulated in cancer cells, such as to drive a suicide gene in cancer therapy.
(b) DescriPtion of Prior Art A successful gene therapy approach is dependent upon two parameters: 1) efficiency of target cells transduction and 2) specificity of gene delivery.
Selective targeting is especially critical in the con-text of cancer therapy for gene directed enzyme prodrug therapy (GDEPT), where a suicide gene expressed in tumor cells encodes an enzyme that converts an other-wise non-toxic prodrug into its active form.
Several methods have been explored to increase the specificity. They can be broadly divided into two categories: directed delivery of the gene of interest or its directed expression. The ideal candidate for transcriptional targeting would be a tumor specific promoter and/or enhancer and its activation will be strong enough to achieve therapeutic levels of the desired transcript. A wide range of promoters have been explored in this context. They were mostly char-acterized as tissue specific promoters as opposed to tumor selective. Some examples are: surfactant protein SP-A promoter for non small cell lung carcinoma (NSCLC), immunoglobulin enhancer or O enhancer for B-cell lineage cancers, tyrosinase for melanomas, and MUC-l/Df3 for breast cancer. However, these promoters also direct gene expression in the normal tissue of origin of these neoplasms and other critical organs as SU~ ITE SHEET (RULE 26) well. The erbB2 and a-fetoprotein promoters are acti-vated to a greater extent in certain neoplasms. They have also been used in this strategy and have lead to promising results. Nonetheless, other promoters to further improve and optimize this strategy are needed.
A striking characteristic of rapidly growing tumor cells is their high rate of glucose utilization compared to their normal counterparts. Glucose is mainly channeled through the glycolytic pathway which is not only used for rapid energy production but also for the provision of biosynthetic precursors necessary to sustain a high rate of cellular division. Hexoki-nase (ATP: D-hexose-6-phosphotransferase) catalyses the first committed step of glycolysis; therefore it was suspected by many to be a potential player in this phe-notype. Hexokinases (HK) are comprised of two highly homologous 5OkDa halves and are product inhibited by glucose-6-phosphate to varying degrees. They exist in four molecular forms, HK I to HK IV, with distinct electrophoretic and kinetic properties (Wilson, J.E., (1985) In Regulation of Carbohydrate Metabolism, Vol I, 45-85, CRC Press, Boca Raton). The profile of these enzymes in tissues at different stages of malignancies shows an increase in HK II in tumor versus normal tis-sues. In rats, the type I HK is expressed in brain,kidney and heart. The type II HK was found in skeletal muscle and in AH130 hepatoma cells. In normal liver it is type IV HK that is most abundant (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (l99S) J. Biol . Chem.
270, 16918-16925).
Comparison of the rat hexokinase II with a hexokinase from rat Novikoff ascites shows there is a single type II isozyme that is found in both normal and tumor tissues (Adams, V., Kempf, W, Hassam, S., and Briner, J. (1995) Biochem. Mol. Med. 54, 53-58). The SUE~STITUTE SHEET (RULE 26) inhibition of HK II by glucose-6-phosphate is delayed.
Therefore, tumors are able to build up high levels of this product. Its accumulation is a signal for glucose availability for consumption, a stimulus of biosyn-thetic pathways for growth (Wilson, J.E., (1985) InRegulation of Carbohydrate Metabolism, Vol I, 45-85, CRC Press, Boca Raton). The level of HK II was also found to be increased in human HepG2 cells and in renal cell carcinoma (Adams, V., Kempf, W, Hassam, S., and Briner, J. (1995) Biochem. Mol. Med. 54, 53-58). Two factors are involved in this increased activity: the propensity of the tumor enzyme to bind to the outer mitochondrial membrane (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem. 270, 16918-16925) and overproduction of the enzyme. The latter isdue to both a gene amplification of the tumor type II
isozyme and to its transcriptional upregulation (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (l99S) J. Biol. Chem. 270, 16918-16925). The promoter for the rat tumor type II enzyme has recently been cloned.
Regulation of the promoter with known modulators of glucose metabolism was found to be different in hepa-toma cells and normal rat hepatocytes (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem.
270, 16918-16925).
It would be highly desirable to be provided with a novel tumor-specific promoter for use in gene tar-geted therapy that is differentially regulated in can-cer cells compared to normal cells.
SU~ ARY OF THE lNVL~ lON
-One aim of the present invention is to provide a novel tumor-specific promoter for use in gene targeted -therapy that is differentially regulated in cancer SU~ JTE SHEET (RULE 26) . . .
cells, such as to drive a suicide gene in cancer ther-apy.
In accordance with the present invention there is provided a tumor-specific promoter for use in gene targeted therapy that is differentially regulated in cancer cells, which comprises Hex II reporter gene.
In accordance with the present invention there is also provided a Hex II gene construct, which com-prises Hex II promoter in a vector selected from pCAT
basic expression vector p~ElsplB and a shuttle plasmid.
In accordance with one embodiment of the present invention the gene construct further comprises ~-gal or HSV Tk.
In accordance with another embodiment of the present invention, the preferred gene construct based on pCAT vector is pHexII4557-CAT.
In accordance with another embodiment of the present invention, the preferred gene constructs based on p~ElsplB are p~ElsplBHex-LacZ and p~ElsplBHex-TK.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l illustrates the Hex II reporter gene con-struct in pCAT basic expression vector in accordance with the present invention;
Fig. 2 illustrates the Hex II promoter construct including ~-galactosidase in the shuttle plasmid pa ElsplB in accordance with the present invention;
Fig. 3 illustrates the Hex II promoter construct including HSV Tk in the shuttle plasmid p~ElsplB in accordance with the present invention;
Fig. 4 illustrates a graph of the results of MUC-l versus HexII promoters activation in normal bron-chial and mAmm~ry epithelial cells; and SU~ ITE SHEET (RULE 26) Fig. 5 illustrates a graph of the results of HexII promoter activation in normal bronchial epithe-lial cells versus non-small cell lung carcinomas.
DETATT~n DESCRIPTION OF THE lNV~..' ION
In accordance with the present invention, there is provided a new HexII promoter. Its constructs are illustrated in Figs. 1 to 3.
1. Construction of recombinant Plasmids pHexII4557-CAT
(8.9 kb) The HexII, 5.15 kb, promoter in the plasmid pUC18 (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem. 270, 16918-16925) was released with an Xbal digest and cloned into the pCAT basic vector (Promega). The size of the promoter was reduced to 4.56 kb with a BamHI digest that released sequences from the non coding region at the 3' end of the clone.
p~ElsplBHex-LacZ
(14.7 kb) the 3.74 kb lacZ gene (HindIII-SalI) from pSV2-~-galactosidase was cloned into the HindIII
and SalI polycloning sites of the shuttle vector p~
ElsplB. This shuttle plasmid contains Adenovirus 5 (Ad5) sequences from map unit 0 to 1, followed by the polycloning site, followed by Ad5 sequences from mu 9.8 to 15.8, and therefore allows recombination to take place with the adenoviral genome. The Hex II promoter 4557 bp was released from the pHexII 4557/CAT with XbaI
followed by an EcoRI digest and cloned into the XbaI
site of the p~ElsplB. Clone 10 (p~ElsplBHexII) that had the insert in the negative orientation relative to the polycloning site of the p~ElsplB was used for fur-ther cloning of the Hex LacZ plasmid. p~ElsplBLacZ was digested with XhoI followed with a partial digest with SU~:~ 111 IJTE SHEET (RULE 26) EcoRI. paElsplBHexII was in turn digested with XhoI
and EcoRI, and the purified 4.6 kb fragment was ligated into p~ElsplBLacZ.
p~ElsplBHex-TK
(12.6 kb) The 1.7 kb HSV-TK gene (EcoRI-SalI) from pMClTK was cloned into the corresponding sites of p~ElsplB. Subsequently, the resulting p~ElsplBTK plas-mid was cut with EcoRI and XhoI, and the purified 4.6 kb HexII fragment with compatible ends was ligated into it. Plasmid DNA was purified by alkaline lysis fol-lowed by cesium chloride density gradient purification.
The use of tissue or tumor selective promoters in targeted gene therapy for cancer depends on strong promoters with specific activity. The Muc-l/Df3 pro-moter has been used in the context of gene directed enzyme prodrug therapy (GDEPT) (Chen et al (1995) J.
Clin. Invest 96(6), 2775). However we have found that it has limited promoter activity and appears to be Z0 expressed in a wide range of normal cells (Fig. 4). An interesting property of cancer cells that could be exploited to target them selectively is their increased rate of glycolysis. Hexokinase type II (Hex II) cata-lyzes the first committed step of glycolysis and has been linked to this phenotype since it is overexpressed in tumors and is not responsive to the normal physio-logical inhibitors, e.g. glucagon (Mathupala, S.P , Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem.
270, 16918-16925).
In accordance with the present invention, the tumor HK II promoter was tested in variety of human tumor cell lines and in normal human cells. We studied the Hex II promoter by transfecting cells with the pHex II4557/CAT (Fig. 1) construct and performing a SUBSTITUTESHEET(RULE26) WO98tl3507 PCT/CA97/00691 chloramphenicol acetyl transferase (CAT) reporter gene assay.
CANCER THERAPY
RACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a novel tumor-specific promoter for use in gene targeted therapy that is dif-ferentially regulated in cancer cells, such as to drive a suicide gene in cancer therapy.
(b) DescriPtion of Prior Art A successful gene therapy approach is dependent upon two parameters: 1) efficiency of target cells transduction and 2) specificity of gene delivery.
Selective targeting is especially critical in the con-text of cancer therapy for gene directed enzyme prodrug therapy (GDEPT), where a suicide gene expressed in tumor cells encodes an enzyme that converts an other-wise non-toxic prodrug into its active form.
Several methods have been explored to increase the specificity. They can be broadly divided into two categories: directed delivery of the gene of interest or its directed expression. The ideal candidate for transcriptional targeting would be a tumor specific promoter and/or enhancer and its activation will be strong enough to achieve therapeutic levels of the desired transcript. A wide range of promoters have been explored in this context. They were mostly char-acterized as tissue specific promoters as opposed to tumor selective. Some examples are: surfactant protein SP-A promoter for non small cell lung carcinoma (NSCLC), immunoglobulin enhancer or O enhancer for B-cell lineage cancers, tyrosinase for melanomas, and MUC-l/Df3 for breast cancer. However, these promoters also direct gene expression in the normal tissue of origin of these neoplasms and other critical organs as SU~ ITE SHEET (RULE 26) well. The erbB2 and a-fetoprotein promoters are acti-vated to a greater extent in certain neoplasms. They have also been used in this strategy and have lead to promising results. Nonetheless, other promoters to further improve and optimize this strategy are needed.
A striking characteristic of rapidly growing tumor cells is their high rate of glucose utilization compared to their normal counterparts. Glucose is mainly channeled through the glycolytic pathway which is not only used for rapid energy production but also for the provision of biosynthetic precursors necessary to sustain a high rate of cellular division. Hexoki-nase (ATP: D-hexose-6-phosphotransferase) catalyses the first committed step of glycolysis; therefore it was suspected by many to be a potential player in this phe-notype. Hexokinases (HK) are comprised of two highly homologous 5OkDa halves and are product inhibited by glucose-6-phosphate to varying degrees. They exist in four molecular forms, HK I to HK IV, with distinct electrophoretic and kinetic properties (Wilson, J.E., (1985) In Regulation of Carbohydrate Metabolism, Vol I, 45-85, CRC Press, Boca Raton). The profile of these enzymes in tissues at different stages of malignancies shows an increase in HK II in tumor versus normal tis-sues. In rats, the type I HK is expressed in brain,kidney and heart. The type II HK was found in skeletal muscle and in AH130 hepatoma cells. In normal liver it is type IV HK that is most abundant (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (l99S) J. Biol . Chem.
270, 16918-16925).
Comparison of the rat hexokinase II with a hexokinase from rat Novikoff ascites shows there is a single type II isozyme that is found in both normal and tumor tissues (Adams, V., Kempf, W, Hassam, S., and Briner, J. (1995) Biochem. Mol. Med. 54, 53-58). The SUE~STITUTE SHEET (RULE 26) inhibition of HK II by glucose-6-phosphate is delayed.
Therefore, tumors are able to build up high levels of this product. Its accumulation is a signal for glucose availability for consumption, a stimulus of biosyn-thetic pathways for growth (Wilson, J.E., (1985) InRegulation of Carbohydrate Metabolism, Vol I, 45-85, CRC Press, Boca Raton). The level of HK II was also found to be increased in human HepG2 cells and in renal cell carcinoma (Adams, V., Kempf, W, Hassam, S., and Briner, J. (1995) Biochem. Mol. Med. 54, 53-58). Two factors are involved in this increased activity: the propensity of the tumor enzyme to bind to the outer mitochondrial membrane (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem. 270, 16918-16925) and overproduction of the enzyme. The latter isdue to both a gene amplification of the tumor type II
isozyme and to its transcriptional upregulation (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (l99S) J. Biol. Chem. 270, 16918-16925). The promoter for the rat tumor type II enzyme has recently been cloned.
Regulation of the promoter with known modulators of glucose metabolism was found to be different in hepa-toma cells and normal rat hepatocytes (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem.
270, 16918-16925).
It would be highly desirable to be provided with a novel tumor-specific promoter for use in gene tar-geted therapy that is differentially regulated in can-cer cells compared to normal cells.
SU~ ARY OF THE lNVL~ lON
-One aim of the present invention is to provide a novel tumor-specific promoter for use in gene targeted -therapy that is differentially regulated in cancer SU~ JTE SHEET (RULE 26) . . .
cells, such as to drive a suicide gene in cancer ther-apy.
In accordance with the present invention there is provided a tumor-specific promoter for use in gene targeted therapy that is differentially regulated in cancer cells, which comprises Hex II reporter gene.
In accordance with the present invention there is also provided a Hex II gene construct, which com-prises Hex II promoter in a vector selected from pCAT
basic expression vector p~ElsplB and a shuttle plasmid.
In accordance with one embodiment of the present invention the gene construct further comprises ~-gal or HSV Tk.
In accordance with another embodiment of the present invention, the preferred gene construct based on pCAT vector is pHexII4557-CAT.
In accordance with another embodiment of the present invention, the preferred gene constructs based on p~ElsplB are p~ElsplBHex-LacZ and p~ElsplBHex-TK.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l illustrates the Hex II reporter gene con-struct in pCAT basic expression vector in accordance with the present invention;
Fig. 2 illustrates the Hex II promoter construct including ~-galactosidase in the shuttle plasmid pa ElsplB in accordance with the present invention;
Fig. 3 illustrates the Hex II promoter construct including HSV Tk in the shuttle plasmid p~ElsplB in accordance with the present invention;
Fig. 4 illustrates a graph of the results of MUC-l versus HexII promoters activation in normal bron-chial and mAmm~ry epithelial cells; and SU~ ITE SHEET (RULE 26) Fig. 5 illustrates a graph of the results of HexII promoter activation in normal bronchial epithe-lial cells versus non-small cell lung carcinomas.
DETATT~n DESCRIPTION OF THE lNV~..' ION
In accordance with the present invention, there is provided a new HexII promoter. Its constructs are illustrated in Figs. 1 to 3.
1. Construction of recombinant Plasmids pHexII4557-CAT
(8.9 kb) The HexII, 5.15 kb, promoter in the plasmid pUC18 (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem. 270, 16918-16925) was released with an Xbal digest and cloned into the pCAT basic vector (Promega). The size of the promoter was reduced to 4.56 kb with a BamHI digest that released sequences from the non coding region at the 3' end of the clone.
p~ElsplBHex-LacZ
(14.7 kb) the 3.74 kb lacZ gene (HindIII-SalI) from pSV2-~-galactosidase was cloned into the HindIII
and SalI polycloning sites of the shuttle vector p~
ElsplB. This shuttle plasmid contains Adenovirus 5 (Ad5) sequences from map unit 0 to 1, followed by the polycloning site, followed by Ad5 sequences from mu 9.8 to 15.8, and therefore allows recombination to take place with the adenoviral genome. The Hex II promoter 4557 bp was released from the pHexII 4557/CAT with XbaI
followed by an EcoRI digest and cloned into the XbaI
site of the p~ElsplB. Clone 10 (p~ElsplBHexII) that had the insert in the negative orientation relative to the polycloning site of the p~ElsplB was used for fur-ther cloning of the Hex LacZ plasmid. p~ElsplBLacZ was digested with XhoI followed with a partial digest with SU~:~ 111 IJTE SHEET (RULE 26) EcoRI. paElsplBHexII was in turn digested with XhoI
and EcoRI, and the purified 4.6 kb fragment was ligated into p~ElsplBLacZ.
p~ElsplBHex-TK
(12.6 kb) The 1.7 kb HSV-TK gene (EcoRI-SalI) from pMClTK was cloned into the corresponding sites of p~ElsplB. Subsequently, the resulting p~ElsplBTK plas-mid was cut with EcoRI and XhoI, and the purified 4.6 kb HexII fragment with compatible ends was ligated into it. Plasmid DNA was purified by alkaline lysis fol-lowed by cesium chloride density gradient purification.
The use of tissue or tumor selective promoters in targeted gene therapy for cancer depends on strong promoters with specific activity. The Muc-l/Df3 pro-moter has been used in the context of gene directed enzyme prodrug therapy (GDEPT) (Chen et al (1995) J.
Clin. Invest 96(6), 2775). However we have found that it has limited promoter activity and appears to be Z0 expressed in a wide range of normal cells (Fig. 4). An interesting property of cancer cells that could be exploited to target them selectively is their increased rate of glycolysis. Hexokinase type II (Hex II) cata-lyzes the first committed step of glycolysis and has been linked to this phenotype since it is overexpressed in tumors and is not responsive to the normal physio-logical inhibitors, e.g. glucagon (Mathupala, S.P , Rempel, A., and Pedersen, P.L. (1995) J. Biol. Chem.
270, 16918-16925).
In accordance with the present invention, the tumor HK II promoter was tested in variety of human tumor cell lines and in normal human cells. We studied the Hex II promoter by transfecting cells with the pHex II4557/CAT (Fig. 1) construct and performing a SUBSTITUTESHEET(RULE26) WO98tl3507 PCT/CA97/00691 chloramphenicol acetyl transferase (CAT) reporter gene assay.
2. Transfection and rePorter qene assays Transient transfections were performed using lipofectamine according to the manufacturer's recommen-dations (GIBCO-BRL). Cells were plated the day before transfection to give 60~ confluency in 6-well plates.
The pl583/+33MUCl.CAT or pHex4557.CAT vectors were transfected along with pSV21acZ to determine promoter activity. 1 ug of each plasmid were used for each well. All conditions assayed were done in duplicate.
The plasmids pRSV.CAT and promoterless pCAT were used as positive and negative controls, respectively. Cells extracts were prepared 48 hours after transfection and ~-galactosidase activity was assayed to compensate for variations in transfection efficiency. CAT activity was determined from 75-100 ug of proteins. The reac-tion was carried out with 0.1 uCi of 14C-labeled chlo-ramphenicol in a 100 ul reaction at 37~C for 4 hrs.
Results Its activation was very high in tumor as opposedto normal cells. The activation of HeX II in the non-small cell lung carcinomas H661 and H460 was 43% and64% (respectively) of the activation observed with the Rous Sarcoma virus (RSV) constitutive promoter while it was 3% of RSV in the primary normal human bronchial epithelial cells (NHBEC). Moreover, treatment of the transfectants with glucagon did non inhibit promoter activation in H661 cells. Its activation in the human ~m~ry carcinoma cells MCF-7 was 72% of RSV while it was 23% of RSV in the normal human m~ m~ry epithelial cells (NHMEC).
SU~ 111 UTE SHEET ~RULE 26) Moreover, the efficacy of this promoter in the context of GDEPT was tested by using the herpes thymid-ine kinase gene in combination with the prodrug gancy-clovir.
The following suicide genes may be used in accordance with the Hex II promoter constructs of the present invention: Cytochrome p_450TM 2Bl with cyclo-phosphamide, penicillin, amidase and ~-lactamase.
The pl583/+33MUCl.CAT or pHex4557.CAT vectors were transfected along with pSV21acZ to determine promoter activity. 1 ug of each plasmid were used for each well. All conditions assayed were done in duplicate.
The plasmids pRSV.CAT and promoterless pCAT were used as positive and negative controls, respectively. Cells extracts were prepared 48 hours after transfection and ~-galactosidase activity was assayed to compensate for variations in transfection efficiency. CAT activity was determined from 75-100 ug of proteins. The reac-tion was carried out with 0.1 uCi of 14C-labeled chlo-ramphenicol in a 100 ul reaction at 37~C for 4 hrs.
Results Its activation was very high in tumor as opposedto normal cells. The activation of HeX II in the non-small cell lung carcinomas H661 and H460 was 43% and64% (respectively) of the activation observed with the Rous Sarcoma virus (RSV) constitutive promoter while it was 3% of RSV in the primary normal human bronchial epithelial cells (NHBEC). Moreover, treatment of the transfectants with glucagon did non inhibit promoter activation in H661 cells. Its activation in the human ~m~ry carcinoma cells MCF-7 was 72% of RSV while it was 23% of RSV in the normal human m~ m~ry epithelial cells (NHMEC).
SU~ 111 UTE SHEET ~RULE 26) Moreover, the efficacy of this promoter in the context of GDEPT was tested by using the herpes thymid-ine kinase gene in combination with the prodrug gancy-clovir.
The following suicide genes may be used in accordance with the Hex II promoter constructs of the present invention: Cytochrome p_450TM 2Bl with cyclo-phosphamide, penicillin, amidase and ~-lactamase.
3. MTT cell viabilitY assaYs Cell survival was determined using a colorimet-ric assay which measures the ability of viable cells to reduce a soluble yellow tetrazolium salt (MTT) to an insoluble purple formazan precipitate. Cells in the logarithmic phase of growth were resuspended at a con-centration of 2x105 cells/ml. 2ml/well were plated in 6-well plates. Plates were incubated for 24 h at 37~C
in 5% CO2. Subsequently, cells were transfected with the p~ElspIB Hex TK plasmid as described above. 6 h after the transfection, cells were treated with the drug gancyclovir at concentrations of lO or 25 ug/ml.
Each condition was done in triplicate. Cells survival was calculated in the treated population as a percent-age of controls. Controls are cells transfected with the plasmid alone or treated with the drug alone. MTT
assays was performed two days following treatment. The formazan crystals were dissolved in dimethyl sulfide (Fisher) and glycine buffer (O.l M glycine- O.l M NaCl, pH lO.5). The formazan product formed by viable cells was quantitated by measuring the absorbance at a wave-length of 570 nm.
Results Cell survival in the transfectants exposed to gancyclovir (GCV) at doses of lO or 25 ug/ml was 50%
SUBSTITUTE SHEET (RULE 26) g less than control cells treated with GCV alone or transfected with the plasmid only. We are presently examining the potential use of Hex II-VTK in recombi-nant Ad5 in the treatment of tumor bearing animals.
The regulation of this promoter in human tumor cell lines was studied using glucose, insulin, and glu-cagon. Lack of metabolic repression was confirmed as described by Mathupala, S.P. et al. ((1995) J. Biol.
Chem. 270, 16918-16925). In addition, several samples of human tissues were screened with the HK I, HK II, and HK IV cDNAs to evaluate the level of these enzymes in tissues and asses the safety of using this promoter in gene therapy.
We hypothesize that the Hex II promoter, with or without the metabolic manipulation of the normally express enzyme in muscle using glucagon will provide and important degree of selectivity to the anti-tumor effect. This represents a novel use of selective pro-moter, taking advantage of its abnormal regulation in tumor cells.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I
In ~ivo 10~A1; zAtion of gene distribution and expression The p~ElspIBHex-LacZ may be used in tumor bear-ing rats for the in vivo localization of the suicide gene in pre-clinical testing of this novel targeting strategy. The gene construct is going to be adminis-tered in adenovirus type 5 recombinant vector or in lipid-based delivery system.
SU~ JTE SHEET (RULE 26) Materials and methods Construction of recombinant viruses Recombinant, replication deficient adenoviral vectors derived from type 5 adenovirus are constructed by the homologous recombination method in the human embryonic kidney cell line 293. The recombinant shut-tle plasmids and pBHGll, containing the adenoviral genome, are co-transfected by calcium phosphate pre-cipitation in 293 cells. The viral DNA is isolated from a single plaque and analyzed by restriction enzyme digestion. Recombinant adenovirus is expanded from a single plaque in 293 cells. Large scale production of the recombinant adenovirus is accomplished by growth in 293 spinner cells and purification by double cesium chloride gradient.
Results These experiments are crucial to determine the best method of administration of the gene construct.
It can either be done regionally to target specific organs such as the liver through portal vein injection or it can be administered intravenously. This method of looking at the distribution of the gene will allow us to determine the efficacy of uptake in the various organs and therefore establish a standard for use in humans.
EXAMPLE II
Targeted gene therapy for suicide destruction of tumors The essential point is that the above-described HexII/VTK construct will be used in a vector/delivery system in clinical trials eventually.
While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications 3~ and this application is intended to cover any varia-SUBSTITUTE SHEET(RULE26) tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
SUBSTITUTE SHEET (RULE 26)
in 5% CO2. Subsequently, cells were transfected with the p~ElspIB Hex TK plasmid as described above. 6 h after the transfection, cells were treated with the drug gancyclovir at concentrations of lO or 25 ug/ml.
Each condition was done in triplicate. Cells survival was calculated in the treated population as a percent-age of controls. Controls are cells transfected with the plasmid alone or treated with the drug alone. MTT
assays was performed two days following treatment. The formazan crystals were dissolved in dimethyl sulfide (Fisher) and glycine buffer (O.l M glycine- O.l M NaCl, pH lO.5). The formazan product formed by viable cells was quantitated by measuring the absorbance at a wave-length of 570 nm.
Results Cell survival in the transfectants exposed to gancyclovir (GCV) at doses of lO or 25 ug/ml was 50%
SUBSTITUTE SHEET (RULE 26) g less than control cells treated with GCV alone or transfected with the plasmid only. We are presently examining the potential use of Hex II-VTK in recombi-nant Ad5 in the treatment of tumor bearing animals.
The regulation of this promoter in human tumor cell lines was studied using glucose, insulin, and glu-cagon. Lack of metabolic repression was confirmed as described by Mathupala, S.P. et al. ((1995) J. Biol.
Chem. 270, 16918-16925). In addition, several samples of human tissues were screened with the HK I, HK II, and HK IV cDNAs to evaluate the level of these enzymes in tissues and asses the safety of using this promoter in gene therapy.
We hypothesize that the Hex II promoter, with or without the metabolic manipulation of the normally express enzyme in muscle using glucagon will provide and important degree of selectivity to the anti-tumor effect. This represents a novel use of selective pro-moter, taking advantage of its abnormal regulation in tumor cells.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I
In ~ivo 10~A1; zAtion of gene distribution and expression The p~ElspIBHex-LacZ may be used in tumor bear-ing rats for the in vivo localization of the suicide gene in pre-clinical testing of this novel targeting strategy. The gene construct is going to be adminis-tered in adenovirus type 5 recombinant vector or in lipid-based delivery system.
SU~ JTE SHEET (RULE 26) Materials and methods Construction of recombinant viruses Recombinant, replication deficient adenoviral vectors derived from type 5 adenovirus are constructed by the homologous recombination method in the human embryonic kidney cell line 293. The recombinant shut-tle plasmids and pBHGll, containing the adenoviral genome, are co-transfected by calcium phosphate pre-cipitation in 293 cells. The viral DNA is isolated from a single plaque and analyzed by restriction enzyme digestion. Recombinant adenovirus is expanded from a single plaque in 293 cells. Large scale production of the recombinant adenovirus is accomplished by growth in 293 spinner cells and purification by double cesium chloride gradient.
Results These experiments are crucial to determine the best method of administration of the gene construct.
It can either be done regionally to target specific organs such as the liver through portal vein injection or it can be administered intravenously. This method of looking at the distribution of the gene will allow us to determine the efficacy of uptake in the various organs and therefore establish a standard for use in humans.
EXAMPLE II
Targeted gene therapy for suicide destruction of tumors The essential point is that the above-described HexII/VTK construct will be used in a vector/delivery system in clinical trials eventually.
While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications 3~ and this application is intended to cover any varia-SUBSTITUTE SHEET(RULE26) tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
SUBSTITUTE SHEET (RULE 26)
Claims (6)
1. A tumor-specific promoter for use in gene targeted therapy that is normally regulated in normal human cells and abnormally regulated in human cancer cells which comprises a Hex II promoter in the negative orientation relative to the polycloning site.
2. A Hex II gene construct, which comprises Hex II
promoter in a vector selected from pCAT basic expression vector p.DELTA.E1sp1B as set forth in fig. 2 and a shuttle plasmid.
promoter in a vector selected from pCAT basic expression vector p.DELTA.E1sp1B as set forth in fig. 2 and a shuttle plasmid.
3. The gene construct of claim 2, which further comprises .beta.-gal or HSV Tk.
4. The gene construct of claim 2, wherein said vector is pCAT and said construct is pHexII4557-CAT as set forth in Fig. 1.
5. The gene construct of claim 3, wherein said vector is p.DELTA.E1sp1B and said construct is p.DELTA.E1sp1BHex-LacZ
as set forth in Fig. 2.
as set forth in Fig. 2.
6. The gene construct of claim 3, wherein said vector is p.DELTA.E1sp1B and said construct is p.DELTA.E1sp1BHex-TK as set forth in Fig. 3.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US2667896P | 1996-09-25 | 1996-09-25 | |
| US60/026,678 | 1996-09-25 | ||
| PCT/CA1997/000691 WO1998013507A1 (en) | 1996-09-25 | 1997-09-22 | Hex ii tumor-specific promoter and uses thereof in cancer therapy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2266846A1 true CA2266846A1 (en) | 1998-04-02 |
Family
ID=21833224
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002266846A Abandoned CA2266846A1 (en) | 1996-09-25 | 1997-09-22 | Hex ii tumor-specific promoter and uses thereof in cancer therapy |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0954590A1 (en) |
| AU (1) | AU4292797A (en) |
| CA (1) | CA2266846A1 (en) |
| WO (1) | WO1998013507A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1130106A1 (en) * | 2000-03-01 | 2001-09-05 | Rijksuniversiteit te Groningen | Non-squamous epithelium-specific transcription |
| AU2003241985A1 (en) * | 2002-05-31 | 2003-12-19 | Medinet Co., Ltd. | Dna inducing cancer cell-specific expression and cancer cell-specific expression vector |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997004104A2 (en) * | 1995-07-14 | 1997-02-06 | The Johns Hopkins University | Tumor type ii hexokinase transcription regulatory regions |
-
1997
- 1997-09-22 EP EP97918865A patent/EP0954590A1/en not_active Withdrawn
- 1997-09-22 WO PCT/CA1997/000691 patent/WO1998013507A1/en not_active Application Discontinuation
- 1997-09-22 AU AU42927/97A patent/AU4292797A/en not_active Abandoned
- 1997-09-22 CA CA002266846A patent/CA2266846A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU4292797A (en) | 1998-04-17 |
| WO1998013507A1 (en) | 1998-04-02 |
| EP0954590A1 (en) | 1999-11-10 |
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