Compositions and methods for inducing gene expression

Details Compositions and methods for inducing gene expression Compositions and methods for inducing gene expression

2000-05-26

2002-08-13

Nguyen, Dave T. (Department: 1632)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S320100, C435S325000, C435S091400, C435S455000, C536S023400, C536S024100, C424S093200

Reexamination Certificate

active

06432927

ABSTRACT:

BACKGROUND OF THE INVENTION
Ischemic heart disease occurs when the heart muscle does not receive an adequate blood supply and is thus deprived of necessary levels of oxygen and nutrients. Ischemia is commonly a result of atherosclerosis which causes blockages in the coronary arteries that provide blood flow to the heart muscle.
Ischemic heart disease can result in certain adaptive responses within the heart which are likely to be beneficial. Among these responses are: 1) increased expression of angiogenic growth factors and their receptors, leading to the formation of collateral circulation around blocked coronary arteries; 2) increased expression of glycolytic enzymes as a means to activate a metabolic pathway which does not require O
2
; and 3) expression of heat shock proteins which can protect the ischemic tissue from death.
At least some of these responses appear to be regulated by a complex oxygen sensing mechanism which eventually leads to the activation of transcription factors which control the expression of critical genes involved in this adaptation. Because this altered gene expression occurs only in response to hypoxia, which usually only occurs when a strain such as exercise is placed upon the diseased heart, cardiac patients do not usually receive much benefit from this endogenous compensatory mechanism. As a result, a number of conventional therapies attempt to supplement the natural therapeutic responses of the heart to ischemia.
For example, such treatments include pharmacological therapies, coronary artery bypass surgery and percutaneous revascularization using techniques such as balloon angioplasty. Standard pharmacological therapy is predicated on strategies that involve either increasing blood supply to the heart muscle or decreasing the demand of the heart muscle for oxygen and nutrients.
Increased blood supply to the myocardium is achieved by agents such as calcium channel blockers or nitroglycerin. These agents are thought to increase the diameter of diseased arteries by causing relaxation of the smooth muscle in the arterial walls. Decreased demand of the heart muscle for oxygen and nutrients is accomplished either by agents that decrease the hemodynamic load on the heart, such as arterial vasodilators, or those that decrease the contractile response of the heart to a given hemodynamic load, such as beta-adrenergic receptor antagonists.
Surgical treatment of ischemic heart disease is based on the bypass of diseased arterial segments with strategically placed bypass grafts (usually saphenous vein or internal mammary artery grafts). Percutaneous revascularization is based on the use of catheters to reduce the narrowing in diseased coronary arteries. All of these strategies are used to decrease the number of, or to eradicate ischemic episodes, but all have various limitations.
More recently, delivery of angiogenic factors or heat shock proteins via protein or gene therapy has been proposed to further augment the heart's natural response to ischemia. Indeed, various publications have discussed the uses of gene transfer for the treatment or prevention heart disease. See, for example, Mazur et al., “Coronary Restenosis and Gene Therapy”,
Molecular and Cellular Pharmacology
21:104-111 (1994); French, B. A. “Gene Transfer and Cardiovascular Disorders”
Herz.
18(4):222-229 (1993); Williams, “Prospects for Gene Therapy of Ischemic Heart Disease”,
Am. J. Med. Sci.
306:129-136 (1993); Schneider and French “The Advent of Adenovirus: Gene Therapy for Cardiovascular Disease”
Circulation
88:1937-42 (1993). International Patent Application No. PCT/US93/11133, entitled “Adenovirus-Mediated Gene Transfer to Cardiac and Vascular Smooth Muscle” reporting the use of adenovirus-mediated gene transfer for regulating function in cardiac vascular smooth muscle.
Accordingly, there exists a need in the art for compositions and methods for inducing the expression of beneficial hypoxia-inducible genes in ischemia-associated cells. Additionally, there exists a need for new vector compositions that allow efficient expression of a range of potentially beneficial genes that are activated by the sustained direct expression of a biologically active mammalian transcription factor. The present invention satisfies these needs and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides recombinant nucleic acid molecules encoding a chimeric transactivator comprising a DNA binding domain of a DNA binding protein wherein the DNA binding protein is a mammalian hypoxia-inducible factor protein, and a functional transcriptional activator domain of a transcriptional activator protein.
Accordingly, in making the invention, we sought to exploit the adaptive response to hypoxia as an alternative approach for the treatment of ischemia associated with vascular disease. We considered that administration of a modified HIF-1&agr; transcription factor via gene therapy might induce expression of a panel of potentially beneficial genes and ultimately lead to the neovascularization of ischemic tissues. We have created a constitutively active form of HIF-1&agr; consisting of the DNA-binding and dimerization domains from HIF-1&agr; and the transactivation domain from herpes simplex virus VP16 protein. Among the possible target genes for this modified transcription factor is VEGF, an endothelial cell-specific mitogen and potent stimulator of angiogenesis.
In vitro analyses of an HIF-1&agr;/VP16 hybrid transcription factor of the invention demonstrated that activation of luciferase reporter constructs under the transcriptional control of either the VEGF or EPO promoters as well as up-regulation of endogenous VEGF gene expression in HeLa and C6 cells was independent of induction. Experiments were performed in a rabbit hindlimb ischemia model to test the hypothesis that exogenous administration of a plasmid encoding HIF-1&agr;/VP16 could enhance collateral vessel formation and also to compare the potency of HIF-1&agr;/VP16 with that of VEGF as an angiogenic therapy. Results of these studies suggest that administration of DNA encoding a transcription factor may represent a viable treatment strategy for tissue ischemia.
The present invention also provides recombinant viral and non-viral vectors that are able to infect and/or transfect and sustain expression of a biologically active chimeric human-viral transactivator protein in mammalian cells.
The present invention further provides a recombinant plasmid vector (pcDNA3/HIF/VP16/Afl2).
In another embodiment, the present invention provides a recombinant plasmid expression vector (pcDNA3/HIF/VP16/R1).
In yet another embodiment, the present invention provides mammalian cells and cell lines transfected with pcDNA3/HIF/VP16/Afl2 or pcDNA3/HIF/VP16/RI.
In still yet another embodiment, the present invention provides recombinant mammalian cell lines able to express biologically active chimeric human-viral transactivator protein at sustained levels.
The present invention also provides recombinant mammalian host cell lines able to express and secrete biologically active chimeric human-viral transactivator protein at sustained levels.
In another embodiment, the present invention provides a fusion protein comprising a DNA binding domain of a DNA binding protein wherein the DNA binding protein is the mammalian hypoxia-inducible factor 1 &agr; (HIF-1&agr;) protein at the amino terminus, and a functional transcriptional activator domain of a transcriptional activator protein, wherein said transcriptional activator protein is HSV VP16 at the carboxy terminus.
The present invention further provides a non-human transgenic mammal expressing recombinant DNA encoding a chimeric transactivator comprising a DNA binding domain of a DNA binding protein wherein said DNA binding protein is the mammalian hypoxia-inducible factor 1 &agr; (HIF-1&agr;) protein, and a functional transcriptional activator domain of a transcriptional activator protein, wherein said transcriptional activator protein is HSV VP16.
In yet another embodiment, the present inv

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