Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-11-03
2004-03-23
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S455000, C435S456000, C435S458000, C424S093200, C514S04400A
Reexamination Certificate
active
06709858
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of gene therapy. More particularly, it concerns methods and compositions for increasing transgene expression.
2. Description of Related Art
Gene therapy now is thought to be widely applicable in the treatment of a variety of cancers and a number of other diseases. Viral vectors are one method employed as a gene delivery system. A great variety of viral expression systems have been developed and assessed for their ability to transfer genes into somatic cells. In particular, retroviral and adenovirus based vector systems have been investigated extensively over a decade. Recently, adeno-associated virus (AAV) has emerged as a potential alternative to the more commonly used retroviral and adenoviral vectors. Lipid vectors including cationic lipids and liposomes also are used to deliver plasmid DNA containing therapeutic genes.
The therapeutic treatment of diseases and disorders by gene therapy involves the transfer and stable or transient insertion of new genetic information into cells. The correction of a genetic defect by re-introduction of the normal allele of a gene encoding the desired function has demonstrated that this concept is clinically feasible (Rosenberg et al.,
New Eng. J. Med.,
323:570 (1990)). Indeed, preclinical and clinical studies covering a large range of genetic disorders currently are underway to solve basic issues dealing with gene transfer efficiency, regulation of gene expression, and potential risks of the use of viral vectors. The majority of clinical gene transfer trials that employ viral vectors perform ex vivo gene transfer into target cells which are then administered in vivo. Viral vectors also may be given in vivo but repeated administration may induce neutralizing antibody.
A major issue facing potential clinical application of gene therapy is the question of how to heterologous genes expressed in clinically significant quantities in selected tissues of the subject. Gene regulatory elements provide a potential answer to that question. Gene regulatory elements such as promoters and enhancers possess cell type specific activities and can be activated by certain induction factors via responsive elements. The use of such regulatory elements as promoters to drive gene expression facilitates controlled and restricted expression of heterologous genes in vector constructs. For instance, heat shock promoters can be used to drive expression of a heterologous gene following heat shock.
U.S. Pat. Nos. 5,614,381, 5,646,010 and WO 89/00603, refer to driving transgene expression using heat shock at temperatures greater than 42° C. These temperatures are not practicable in human therapy as they can not be maintained for a sustained period of time without harm to the individual.
Gene therapy could be used in combination with a variety of conventional cancer therapy treatments including cytotoxic drugs an radiation therapies. It has been shown that hyperthermia enhances the cell killing effect of radiation in vitro (Harisiadis et al.,
Cancer,
41:2131-2142 (1978)), significantly enhances tumor response in animal tumors in vivo and improves the outcome in randomized clinical trials. However, the major problem with the use of hyperthermia treatment is that the hyperthermia system can not adequately heat large and deep tumors.
Thus, it would be useful to develop vectors that may be used at temperatures of 42° C. and below, systemically or locally, to treat a patient such that the expression of the therapeutic gene(s) is activated preferentially in regions of the body that have been subjected to conditions which induce such expression.
SUMMARY OF THE INVENTION
The present invention provides methods for effecting the inducible expression of polynucleotides in cells. In particular, the use of heat shock promoters in methods for effecting the inducible expression of polynucleotides in mammalian cells is taught. The present invention overcomes deficiencies in the prior art by providing heat shock-controlled vectors that may be used at temperatures of 42° C. and below. These methods may be used to treat a patient via the inducible expression of a therapeutic gene.
In one embodiment, the present invention provides a method for effecting transgene expression in a mammalian cell that comprises first providing an expression construct that comprises both (i) an inducible promoter operably linked to a gene encoding a transactivating factor and (ii) a second promoter operably linked to a selected polynucleotide. The second promoter is activated by the transactivating factor expressed by the same construct. The method then includes the step of introducing the expression construct into the cell. Finally, the cell is subjected to conditions which activate the inducible promoter and result in the expression of the selected polynucleotide.
In a preferred embodiment of the invention, the inducible promoter is a heat shock promoter and the conditions which activate the heat shock promoter are hyperthermic conditions. The hyperthermic conditions may comprise a temperature between about basal temperature and about 42° C. As used herein the basal temperature of the cell is defined as the temperature at which the cell is normally found in its natural state, for example, a cell in skin of a mammal may be at temperatures as low as 33° C. whereas a cell in the liver of an organism may be as high as 39° C. In specific embodiments, the application of hyperthermia involves raising the temperature of the cell from basal temperature, most typically 37° C. to about 42° C. or less. Alternatively, the hyperthermic conditions may range from about 38° C. to about 41° C., or from about 39° C. to about 40° C. The heat shock promoter is optionally derived from a promoter selected from the group of the heat shock protein (HSP) promoters HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25 and HSP28. The ubiquitin promoter may also be used as the heat-shock inducible promoter in the expression construct. A minimal heat shock promoter derived from HSP70 an comprising the first approximately 400 bp of the HSP70B promoter may optionally be used in the invention.
In an alternative embodiment, the inducible promoter comprises a hypoxia-responsive element (HRE). This hypoxia-response element may optionally contain at least one binding site for hypoxia-inducible factor-1 (HIF-1).
In one embodiment of the invention, the second promoter may be selected from the group consisting of an human immunodeficiency virus-1 (HIV-1) promoter and a human immunodeficiency virus-2 (HIV-2) promoter. In preferred embodiments, the transactivating factor may be a transactivator of transcription (TAT).
The selected polynucleotide may code for a protein or a polypeptide. For instance, the selected polynucleotide may encode any one of the following proteins: ornithine decarboxylase antizyme protein, p53, p16 , neu, interleukin-1 (IL1), interleukin-2 (IL2), interleukin-4 (IL4), interleukin-7 (IL7), interleukin-12 (IL12), interleukin-15 (IL15), FLT-3 ligand, granulocyte-macrophage stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), gamma-interferon (INF&ggr;), alpha-interferon (IFN&agr;), tumor necrosis factor (TNF), herpes simplex virus thymidine kinase (HSV-TK), I-CAM1, human leukocyte antigen-B7 (HLA-B7), or tissue inhibitor of metalloproteinases (TIMP-3). In such an embodiment, the selected polynucleotide is positioned in a sense orientation with respect to the second promoter.
Alternatively, expression of the selected polynucleotide may involve transcription but not translation and produces a ribozyme. In this embodiment, the selected polynucleotide is also positioned in a sense orientation with respect to the second promoter.
In still another alternative embodiment, the expression of the selected polynucleotide involves transcription but not translation and results in an RNA molecule which serves as an antisense nucleic acid. In such an embodiment, the selected polynucleotide may be the target gene, or a fr
Gerner Eugene W.
Harris David T.
Hersh Evan
Tsang Tom
Priebe Scott D.
The Arizona Board of Regents on behalf of The University of Ariz
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