Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-02-22
2001-07-24
Martin, Jill D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S455000, C435S320100, C435S325000, C435S069100, C424S093210
Reexamination Certificate
active
06265390
ABSTRACT:
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).
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.
SUMMARY OF THE INVENTION
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 targetted 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.
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Firth John David
Harris Adrian Llewllyn
Pugh Christopher William
Ratcliffe Peter John
Stratford Ian James
Martin Jill D.
Oxford Biomedica (UK) Limited
Wenderoth , Lind & Ponack, L.L.P.
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