Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-08-17
2004-07-06
Shukla, Ram R. (Department: 1635)
Chemistry: molecular biology and microbiology
Vector, per se
C435S006120, C435S091100, C435S091400, C536S023100, C536S024500
Reexamination Certificate
active
06759236
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of gene therapy for cancer. More specifically, the present invention presents a method of controlling the expression of therapeutically valuable gene products via inducible promoters. The present invention provides a method whereby induced gene expression in the intended cell targets is enhanced and prolonged in a spatially and temporally regulable manner by means of heat or light inducible promoters. Moreover, the present invention provides a method whereby the background gene expression in non-targeted cells is reduced or eliminated.
2. Description of the Related Art
One of the major obstacles to the success of chemotherapy and radiation therapy for cancer is the difficulty in achieving tumor-specific cell killing. The inability of radiation or cytotoxic chemotherapeutic agents to distinguish between tumor cells and normal cells necessarily limits the dosage that can be applied. As a result, diseases relapse due to residual surviving tumor cells is frequently observed.
The use of gene therapy in cancer treatment presents many of the same disadvantages as chemotherapy and radiation therapy. Problems with current state-of-the-art gene therapy strategies include the inability to deliver the therapeutic gene specifically to the target cells. This leads to toxicity in cells that are not the intended targets. For example, manipulation of the p53 gene suppresses the growth of both tumor cells and normal cells, and intravenous administration of tumor necrosis factor alpha (TNF&agr;) induces systemic toxicity with such clinical manifestations as fever and hypertension.
Attempts have been made to overcome these problems. These include such strategies as: the use of tissue-specific receptors to direct the genes to the desired tissues (Kasahara, N., et al.,
Science
, 266:1373-1376 (1994)), the use of tissue-specific promoters to limit gene expression to specific tissues (e.g. use of the prostate specific antigen promoter) and the use of heat (Voellmy R., et al.,
Proc. Natl. Acad. Sci. USA
, 82:4949-4953 (1985)) or ionizing radiation inducible enhancers and promoters (Trainman, R. H., et al.,
Cell
46: 567-574 (1986); Prowess, R., et al.,
Proc. Natl. Acad. Sci. USA
85, 7206-7210 (1988)) to enhance expression of the therapeutic gene in a temporally and spatially controlled manner. The heat inducible heat shock protein (HSP) promoter has been used to direct the expression of genes such as the cytokine IL-2.
Weichselbaum and colleagues were the first to discover the radiation inducible response of the early growth response (Egr-1) gene promoter. Accordingly, they have attempted to direct expression of such cytotoxic genes as TNF-&agr; to tumor cells to enhance radiation cell killing by means of this promoter. Previously, systemic administration of the cytokine TNF-&agr; as an adjuvant to ionizing radiation was initially reported to result in enhanced killing in a mouse xenograft tumor system. It has since been shown partially effective in human tumors. The effect of TNF&agr; appears to be dosage-dependent, as its tumor-killing effect correlates with its serum concentration. However, systemic toxicity of TNF&agr; restricts the dosage that can be applied and thus limits the usefulness of the treatment regimen. Attempts have also been made to deliver the TNF&agr; gene to tumor cells via adenoviral vector and/or liposomes. Unfortunately, expression of the TNF&agr; gene is not restricted to the tumor sites due to the ‘leakiness’ of the promoter.
In an attempt to localize the level of TNF&agr; to the general area of radiation exposure and thereby reduce systemic toxicity, Weichselbaum and colleagues employed the radiation inducible Egr-1 promoter to activate the TNF&agr; gene in situ. Earlier studies showed that the expression of certain immediate-early genes such as jun/fos and Egr-1 are activated in cells exposed to ionizing radiation (Sherman, M. L., et al.,
Proc. Natl. Acad. Sci. USA
, 87: 5663-5666 (1997); Hallahan, D. E., et al.,
Proc. Natl. Acad. Sci. USA
, 88: 2156-2160 (1991)). By placing the TNF&agr; gene under the control of the Egr1 promoter (EGRp), the expression of the TNF&agr; is enhanced in those cells harboring an EGRp-TNF&agr; plasmid when exposed to ionizing radiation. In vivo, the serum level of TNF&agr; is greatly enhanced (Weichselbaum R. R., et al.,
Cancer Res.
54: 4266-4269 (1994)) within a few hours after irradiation. The combined treatment with this plasmid and radiation leads to a partial regression of a xenografted tumor during the course of the treatment. The level of TNF&agr; dropped precipitously within 24 hours; further decreases in serum level of TNF&agr; coincided with regrowth of the tumors.
There are several possible reasons for the recurrence of the tumor upon cessation of therapy. The most obvious reason is probably the same limitation seen with chemotherapy or radiation therapy in general, viz., insufficient dosage levels. A major problem, which limits the amount of TNF&agr; produced, is the weak and transient nature of the Egr-1 promoter. This promoter is intrinsically weak, with a maximum of less than three-fold increase in expression upon induction. Moreover, the induced expression is of necessity transient. This, coupled with the weakness of the promoter, permits only a brief exposure of the tumor cells to the TNF&agr;.
Another factor that limits the production of sufficient dosage of TNF&agr; is that not every tumor cell will have taken up the TNF&agr; plasmid. While it has been suggested that repeated administration may help to improve the treatment outcome, it is not clear if the repeated delivery of a suboptimal low dosage of TNF&agr; will be useful, the problems posed by an immune response notwithstanding. Although it might be conceivable to deliver larger doses of plasmids, the problem of promoter leakiness has hindered such an approach. It is known that a substantial basal level of activity (20-30%) can be detected with the Egr-1 promoter even in the absence of ionizing radiation (Weichselbaum, et al., supra). This is not surprising, as the radiation response element, a CArG box, is part of the serum response element.
The HSP promoter is also rather leaky. In the absence of heat, this promoter exhibits a 25-30% background level of expression, not suitable for most cytotoxic genes. As this level of expression will be harmful to unirradiated normal cells that take up the gene. Hence, administration of this plasmid has been restricted to small doses of intra-tumoral injections to minimize systemic toxicity.
Therefore, while it may be advantageous to employ a spatially and temporally regulated promoter such as the HSP and Egr-1 promoters to enhance specificity of gene expression at the site of heat or radiation treatment, current versions of those promoters have serious problems that restrict their applicability. In order to apply these promoters for use in cancer therapy, it is necessary to eliminate or greatly reduce background expression in unheated or unirradiated cells. Ideally, the expression of cytotoxic genes should be limited to the area of external stimuli (heat or radiation). Additionally, to ensure a sufficient level of expression of therapeutic genes, the weak and transient nature of gene expression driven by these promoters must be improved.
It is important to note that even when an improved inducible vector system which can restrict the expression of a therapeutic gene to the area of external stimuli is developed, there is still the problem of expression in normal heated or irradiated bystander cells. Thus, it is critical to be able to further restrict the expression of therapeutic genes only to the intended targets, e.g., tumor cells.
The prior art is deficient in the lack of effective means of inhibiting unwanted toxic side effects of gene therapy treatments for cancer, as well as providing a method for enhancing and sustaining gene expression in targeted tumor cells in a controllable manner. The present invention fulfi
Fung Yuen Kai
Gomer Charles
T'Ang Anne
Adler Benjamin Aaron
Research Development Foundation
Shukla Ram R.
Zara Jane
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