| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 24, 1999 |
| PATENT TITLE |
Nucleic acid constructs comprising hypoxia response elements |
| PATENT ABSTRACT | Nucleic acid constructs comprising hypoxia response elements in operable linkage with a coding sequence of a gene of interest are disclosed. In particular, such nucleic acid constructs comprise genes encoding pro-drug activation systems or cytokines |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 12, 1996 |
| PATENT CT FILE DATE | February 15, 1995 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | August 17, 1995 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
K. Blanchard et al. "Hypoxic Induction of the Human Erythropoietin Gene: Cooperation Between the Promoter and Enhancer, Each of Which Contains Steroid Receptor Response Elements", Molecular and Cellular Biology, vol. 12, No. 12, pp. 5373-5385, Dec. 1992. C. Bauer, "Erythropoietin- From Gene Structure to Therapeutic Applications", J. Perinat. Med. vol. 23, p. 77-81, 1995. S. Imagawa et al., "Regulatory elements of the erythropoietin gene", Blood, vol. 77, pp. 278-285, Jan. 15, 1991. P. Maxwell et al., "Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: Evidence for a widespread oxygen-sensing mechanism", Proc. Natl. Acad. Sci., vol. 90, pp. 2423-2427, Mar. 1993. R. Vile et al., "In Vitro and in vivo targeting of gene expression to melanoma cells", Cancer Research, vol. 53, pp. 962-967, Mar. 1, 1993. B. Lim et al., "Retrovirus-mediated gene transfer of human adenosine deaminase: Expression of functional enzyme in murine hematopoietic stem cells in vivo", Mol. Cell. Biol., vol. 7, No. 10, pp. 3459-3465, Oct. 1987. G. Semenza et al., "Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene", Proc. Natl. Acad. Sci., vol. 88, pp. 5680-5684, Jul. 1991. C. Pugh et al., "Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene", Proc. Natl. Acad. Sci., vol. 88, pp. 10553-10557, Dec. 1991. Eck & Wilson, `Gene-Based Therapy.` In: Goodman's & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill Health Professions Division, Chapter 5, pp. 77-101, 1995. Dachs et al., Nature Medicine, vol. 3, No. 5, pp. 515-520, May 1997. Orkin et al., Report and Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy, Dec. 7, 1995. Hanania, American Journal of Medicine, vol. 99, No. 5, p. 537-552, Nov. 1995. Mullen et al., Proceedings of the National Academy of Sciences of the USA, vol. 89, No. 1, pp. 33-37, Jan. 1, 1992. |
| PATENT CLAIMS |
We claim: 1. A nucleic acid construct comprising a hypoxia response element, wherein said hypoxia response element is a phosphoglycerate kinase sequence between about nucleotide -523 to about nucleotide -21 of the mouse PGK-1 gene which is nucleotide 423 to nucleotide 925 of SEQ ID No. 4, or a hypoxia-responsive fragment thereof, operably linked to a coding sequence other than the mouse PGK-1 coding sequence. 2. The nucleic acid construct according to claim 1, wherein the hypoxia-responsive fragment comprises the nucleic acid sequence of SEQ ID No. 2. 3. The nucleic acid construct according to claim 1, wherein the hypoxia-responsive fragment comprises the nucleic acid sequence of SEQ ID No. 3. 4. The nucleic acid construct according to claim 1, which further comprises an additional one or more of said hypoxia response elements. 5. A nucleic acid construct comprising a hypoxia response element, wherein said hypoxia response element is a lactate dehydrogenase sequence between about nucleotide -186 to about nucleotide +47 of the mouse LDH-A gene which is nucleotide 932 to nucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragment thereof, operably linked to a coding sequence other than the mouse LDH-A coding sequence. 6. The nucleic acid construct according to claim 5, which further comprises an additional one or more of said hypoxia response elements. 7. A nucleic acid construct comprising two or more hypoxia response elements, wherein said hypoxia response elements consist of two or more copies of the nucleic acid sequence of SEQ ID NO. 1, operably linked to a coding sequence. 8. A method for preparing a nucleic acid construct according to claim 1, comprising the steps of: (a) providing a hypoxically inducible expression control sequence between about nucleotide -523 to about nucleotide -21 of the mouse PGK-1 gene which is nucleotide 423 to nucleotide 925 of SEQ ID No. 4, or a hypoxia-responsive fragment thereof; (b) providing a nucleic acid sequence encoding a gene product, other than mouse PGK-1; and (c) operably linking the hypoxically inducible expression control sequence to the nucleic acid sequence. 9. The method according to claim 8, wherein the hypoxia-responsive fragment comprises the nucleic acid sequence of SEQ ID No. 2. 10. The method according to claim 8, wherein the hypoxia-responsive fragment comprises the nucleic acid sequence of SEQ ID No. 3. 11. The method according to claim 8, wherein the gene product is an enzyme which converts a prodrug to an active drug. 12. The method according to claim 11, wherein the enzyme is cytosine deaminase. 13. The method according to claim 8, wherein the gene product is a cytokine. 14. A method for preparing a nucleic acid construct according to claim 5, comprising the steps of: (a) providing a hypoxically inducible expression control sequence between about nucleotide -186 to about nucleotide +47 of the mouse LDH-A gene which is nucleotide 932 to nucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragment thereof; (b) providing a nucleic acid sequence encoding a gene product, other than mouse LDH-A; and (c) operably linking the hypoxically inducible expression control sequence to the nucleic acid sequence. 15. The method according to claim 14, wherein the gene product is an enzyme which converts a prodrug to an active drug. 16. The method according to claim 15, wherein the enzyme is cytosine deaminase. 17. The method according to claim 15, wherein the gene product is a cytokine. 18. A method for preparing a nucleic acid construct according to claim 7, comprising the steps of: (a) providing two or more hypoxically inducible expression control sequences which consist of two or more copies of the nucleic acid sequence of SEQ ID No. 1; (b) providing a nucleic acid sequence encoding a gene product; and (c) operably linking the hypoxically inducible expression control sequences to the nucleic acid sequence. 19. The method according to claim 18, wherein the gene product is an enzyme which converts a prodrug to an active drug. 20. The method according to claim 19, wherein the enzyme is cytosine deaminase. 21. The method according to claim 18, wherein the gene product is a cytokine. 22. An isolated DNA consisting of SEQ ID No. 2 or 3. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention This invention is concerned with hypoxically-inducible expression control sequences, nucleic acid constructs comprising such sequences, and their use for selective targeting of anti-cancer therapy and other kinds of therapy where target cells are affected by hypoxia. 2. Detailed Description of the Related Art Vile and Hart (1993) describe a method in which certain gene promoters, which are preferentially active in melanocytic cells, were used to direct gene expression of a reporter gene specifically to melanoma cells in vitro, and in vivo in mice. Constructs consisting of the promoters and the beta-galactosidase gene were directly injected into mice and the reporter gene was expressed in melanoma cells and in some normal melanocytes but not in surrounding normal tissue. However, tissue-specific promoters will necessarily be limited in the tumours that they can target and will also be liable to target normal cells of the tissue concerned (as was noted in Vile and Hart above). SUMMARY OF THE INVENTION Cancers tend to outgrow the blood supply and often have areas of hypoxia and necrosis which distinguish them from normal tissue. This feature also makes tumours resistant to radiation due to low oxygen levels and x-ray treatment becomes less effective. Certain genes such as the gene for erythropoietin, are known to be regulated by hypoxia. Erythropoietin is a hormone which regulates erythropoiesis and hence blood oxygen content. Cis-activating DNA sequences that function as tissue-specific hypoxia-inducible enhancers of human erythropoietin expression have been identified (Semenza et al, 1991). A DNA enhancer sequence located 3' to the mouse erythropoietin gene has been shown to confer oxygen-regulated expression on a variety of heterologous promoters (Pugh et al, 1991). It has further been demonstrated that the oxygen-sensing system which controls erythropoietin expression is widespread in mammalian cells (Maxwell et al, 1993). A second example of a hypoxia-associated regulator is a regulator which lies 5' to the mouse phosphoglycerate kinase gene promoter. The sequence of the regulator has been published (McBurney et al, 1991) but its hypoxia inducible properties have not previously been considered or defined in the literature. It has now been recognised by the inventors that the native sequence of the regulator has hypoxically-inducible features. The nucleotides responsible have been defined and the inventors have shown that repeating the sequence leads to increased induction of the gene whose expression is controlled. Further, the inventors have shown that using the interleukin-2 gene under tissue-specific promoters is an effective strategy for specific targeting of tumours. There are anti-cancer drugs that become activated under hypoxia (Workman and Stratford, 1993), but the use of a drug activation system where the enzyme activating the drugs is greatly increased under hypoxia will provide a far superior therapeutic effect. The invention provides a nucleic acid construct comprising at least one gene encoding a species having activity against disease, operatively linked to a hypoxically inducible expression control sequence. When the construct is present in a suitable host cell, expression of the gene will thus be regulated according to the level of oxygenation. Preferably the expression control sequence is a promoter or enhancer. In a host cell under hypoxic conditions, expression of the gene will be initiated or upregulated, while under conditions of normoxia (normal oxygen level) the gene will be expressed at a lower level or not expressed at all. The expression level may vary according to the degree of hypoxia. Thus, a gene product which has therapeutic activity can be targeted to cells affected by disease, eg. tumour cells. The species encoded by the gene in the construct according to the invention may be for example a cytokine, such as interleukin-2 (IL-2) which is known to be active in the immune response against tumours. Genes encoding other molecules which have an anti-tumour effect may also be used. In a preferred embodiment of the construct according to the invention, the species encoded by the gene is a pro-drug activation system, for example the thymidine phosphorylase enzyme, which converts a relatively inactive drug into a much more potent one. Transfection of the thymidine phosphorylase gene into human breast cancer cells has been shown to greatly increase the sensitivity of the cancer cells to 5-deoxy-5FU (see Example 8). The thymidine phosphorylase gene has not previously been reported as an agent for gene therapy. Another pro-drug activation system which can be used is cytosine deaminase, which activates the pro-drug 5-fluorocytosine (5-FC) to form the antitumour agent 5-fluorouracil (5-FU). A further example of a pro-drug activation system for use in the invention is cytochrome p450 to activate the drug SR4233 (Walton et al, 1992). The construct according to the invention may contain more than one gene and more than one type of gene. Additional genes may encode further species having activity against disease, or they may have gene products with other activities. DESCRIPTION OF THE DRAWINGS FIGS. 1A-1B. FIG. 1A shows the structure of the test plasmids, where E represents Epo enhancer sequence, SV40P represents the SV40 early promoter and GH represents the body of the growth hormone gene. FIG. 1B shows the independent experiments measuring hypoxic inducible activity of the constructs comprising the Epo enhancer sequence. FIGS. 2A-2B. FIG. 2A shows the partial nucleotide sequence of the 253 bp EcoR1-Spe1 fragment containing the enhancers (EMBL accession No. M18735, nucleotides 417 to 676). The position of the deletions (D) from the 5' end of the sequence are marked by arrows. FIG. 2B shows the ratio of hypoxic to normoxic expression conferred on the reporter by EcoR1-Spe1 fragment and deletion thereof. Values represent means of three separate transfections, with bars showing SEM. FIG. 3 shows a survival curve for the parent cell line MCF-7 in breast cancer (WT) and a derivative from it that has been transfected with a gene for thymidine phosphorylase, MCF-7(-4). FIG. 4 shows the drug sensitivity results of transfectant (-4) to 5-deoxy 5FUdR as compared to the wild-type. FIG. 5 shows the drug sensitivity results of clones -4, -7, -12, -16, and +4 to 5-deoxy 5FUdR compared to the wild-type. FIG. 6 shows the drug sensitivity results of clones -4, -7, -12, -16, and +4 to 5FU. FIG. 7 shows the drug sensitivity results of the mixed cell population (-4:wt) to 5-deoxy 5FUdR. FIGS. 8A-8C. FIG. 8A shows CD2 induction data for M3. FIG. 8B shows CD2 induction data for sTK5. FIG. 8C shows CD2 induction data for 9-3C. In FIGS. 8A-8C, the CD2 induction data was measured with oxygen at 5%, 2%, 1%, 0.001% (N.sub.2), almost no oxygen), 0% O.sub.2 (AnO.sub.2). n represents the number of experiments performed. CD2 expression was measured immediately following treatment (Oh) and five hours post-treatment (5 h). FIGS. 9A-9B. FIG. 9A shows drug sensitivity results of transfected cells 9-3C and M3 to 5-FU post-hypoxia. FIG. 9B shows drug sensitivity results of transfected cells 9-3C and M3 to 5-FC post-hypoxia. Hypoxically-inducible promoters or enhancers may be chosen from those referred to herein, or they may be other hypoxically-inducible promoters or enhancers. It is anticipated that other hypoxically-inducible promoters or enhancers will be discovered; oxygen-sensing systems are widespread in mammalian cells and many genes are likely to be under hypoxic control. Preferably, the nucleic acid construct according to the invention comprises at least one hypoxia response element which confers hypoxia inducibility on the expression control sequence. There may be for example two or more hypoxia response elements linked so as to increase hypoxia inducibility and thus to increase the induction of the gene or genes under hypoxia. Hypoxia response elements may be chosen from among those referred to herein, or they may be other hypoxia response elements. As noted above, oxygen-sensing systems are widespread in mammalian cells, and it is expected that other hypoxia response elements will be found. The following hypoxia response elements may be used in the construct according to the invention: a). The following portion of the transcriptional enhancer lying 3' to the mouse erythropoietin (Epo) gene: GGG CCC TAC GTG CTG CCT CGC ATG G (25) ›SEQ ID NO: 1! b) One of the following portions of the 5' flanking sequence of the mouse phosphoglycerate kinase (PGK) gene: CGC GTC GTG CAG GAC GTG ACA AAT (P24) ›SEQ ID NO: 2! or GTC GTG CAG GAC GTG ACA (P18) ›SEQ ID NO: 3! These PGK sequences have not been previously recognised as having hypoxically-inducible properties. All of these sequences have counterparts in human genes and are highly conserved between species. They are also well characterised. The invention therefore also provides a hypoxically inducible expression control sequence which comprises the nucleic acid sequence: CGC GTC GTG CAG GAC GTG ACA AAT (P24) ›SEQ ID NO: 2! or GTC GTG CAG GAC GTG ACA (P18) ›SEQ ID NO: 3! or a nucleic acid sequence with substantial homology thereto. These sequences can be found in EMBL database, accession no. M18735, at nucleotides 631 to 654 and 634 to 651. The construct according to the invention may comprise more than one eg. three or more copies of one of the Epo or PGK sequences given above. Additionally or alternatively, a longer portion of the Epo or PGK-1 enhancer or flanking sequence may be used in the construct, which longer portion comprises the hypoxia response element and part of the surrounding sequence. Hypoxically-inducible expression control sequences and hypoxia response elements may be chosen so as to be operative in particular tissues or cell types to be targeted therapeutically, or they may be chosen to work in a wide range of tissues or cell types. The invention further provides a nucleic acid construct as described herein for use in the treatment of a patient suffering from a disease in which hypoxia is a cause or a symptom or is otherwise present. Alternatively, the invention provides a method of treatment of a patient suffering from a disease in which hypoxia is a cause or a symptom or is otherwise present, which method comprises administering to the patient a nucleic acid construct as described herein. The nucleic acid constructs will be useful for treating cancer patients. In hypoxic tumour cells, the physiological stimuli will be such that the gene which has activity against the disease is expressed. Although it may be very difficult to get all cells to take up the gene to switch it on, experiments show that even with 10% of cells expressing the thymidine phosphorylase gene there is an effect on other cells which results in a 10-fold increase in sensitivity of the whole population. This is known as the bystander effect and is probably due to active metabolites of the anticancer drug passing from one cell to another. Since this drug kills proliferating cells, it should still have much less toxicity on normal tissue than on cancer cells. Sometimes, nearby normal cells will also express the gene and the active drug or cytokine or other species encoded by the gene will be able to diffuse from the normal cells into the tumour. The inventors have demonstrated both stable and transient transfection of cells with genes under the control of hypoxically inducible enhancers. Transient transfection only lasts for a few days, whereas stably transfected genes insert into the cell genome and can persist indefinitely. Stable transfection may prove to be necessary for therapeutic applications of the invention but it is possible that transient transfection will be sufficient. Administration of constructs according to the invention for therapeutic purposes can be by injection of DNA directly into the solid tumour. Certain types of cell including tumour cells, skin cells and muscle cells take up naked DNA, and tumour cells do so particularly well. The constructs may thus be administered in the form of naked DNA plasmids. Alternatively other vectors such as retroviruses may be used. A suitable therapeutic regime will be direct injection of DNA into the affected site, and administration of pro-drug in the case of a construct encoding a pro-drug activation system, optionally combined with radiotherapy. Although the invention is described above in relation to targeting of tumour cells, it will also be useful in other types of disease where hypoxia occurs, including for example coronary artery disease and strokes. The nucleic acid construct may comprise a gene encoding a pro-drug activation system for a suitable drug or the gene may encode a cytokine or a growth factor. A vascular growth factor can be used to stimulate new blood vessel formation in hypoxic areas and expression of the growth factor by the construct will then be automatically switched off when the area becomes revascularised. The inventors have carried out deletional analysis and mutational analysis of the mouse Epo enhancer sequence, in the cell lines HepG2 and a23 (a23 are non-Epo producing cells). Transient tranfection experiments were performed using plasmids containing full or partial enhancer sequences lying 1.4 kb 5' to the .alpha..sub.1 globin reporter gene. Three critical sites for enhancement were defined, corresponding to the nucleotides 5-12, 21-24 and 33-34 of the 96 nucleotide enhancer sequence (mouse Epo enhancer: EMBL accession no. X73471). All three regions were absolutely necessary for enhancer function in the above experiments. But overall, studies indicated that the 1-25 or 1-26 nucleotide sequence has hypoxically inducible operation (EMBL, X73471, nucleotides 407 to 431 or 432. Inducible operation via the 1-26 nucleotide sequence was also demonstrated for MEL and HeLa cells, which have previously been found unable to support oxygen-regulated reporter gene expression using plasmids containing the 1-96 nucleotide enhancer sequence (Maxwell et al 1993). A total of 19 cell lines (some unreported) have now been tested by the inventors and none has been found to lack the capacity to modulate oxygen dependent changes in reporter gene expression by the sequence 1-26. Thus, whereas it was previously suggested that the hypoxically inducible response was not universal in mammalian cells, it now seems more likely that it is. Recent studies of the human Epo enhancer also define at least three critical regions of the sequence (Semenza et al 1992 and Blanchard et al 1992). The examples which follow contain experimental demonstrations by the inventors which indicate the manner in which different aspects of the invention work. |
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| PATENT PHOTOCOPY | Available on request |
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