Compositions and methods for inducing gene expression

a technology of inducible gene expression and composition, applied in the direction of drug composition, cardiovascular disorder, peptide, etc., can solve the problems of deprived of necessary levels of oxygen and nutrients, cardiac patients do not usually receive much benefit from this endogenous compensatory mechanism, and all have various limitations. , to achieve the effect of increasing the expression of hypoxia-inducible genes

Inactive Publication Date: 2006-06-15
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] In vitro analyses of an HIF-1α / 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-1α / VP16 could enhance collateral vessel formation and also to compare the potency of HIF-1α / 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.
[0017] 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.
[0018] 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.
[0021] In yet another embodiment, the present invention provides a method for increasing expression of hypoxia-inducible genes.
[0022] In still yet another embodiment, the present invention provides a method for providing sustained expression of biologically active HIF-1α under normoxic conditions.

Problems solved by technology

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.
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.
All of these strategies are used to decrease the number of, or to eradicate ischemic episodes, but all have various limitations.

Method used

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  • Compositions and methods for inducing gene expression
  • Compositions and methods for inducing gene expression
  • Compositions and methods for inducing gene expression

Examples

Experimental program
Comparison scheme
Effect test

example 1

Hybrid / Chimera Construction

[0119] A hybrid transcription factor (pcDNA3 / HIF.VP-16.Afl2) composed of a DNA binding and dimerization domains from HIF-1α and the transactivation domain from herpes simplex virus VP16 (FIG. 1) was constructed to provide strong, constitutive activation of genes normally involved in the physiological adaptation to hypoxia. As is described below, we analyzed the effect of this HIF-1α / VP16 transcription factor on VEGF gene expression in vitro, and on neovascularization in a rabbit hindlimb ischemia model.

Recombinant Plasmids

[0120] The fill-length (aa1-826) HIF-1α gene was isolated by PCR (Advantage cDNA PCR Kit, Clontech, Palo Alto, Calif.) from a HeLa cell cDNA library (Clontech) using the primers set forth in SEQ ID NO's 1 and 2 and inserted between the KpnI and XbaI sites of the expression vector, pcDNA3 (Invitrogen, Carlsbad, Calif.). In this plasmid, gene expression is controlled by the cytomegalovirus (CMV) immediate early enhancer / promoter. The HI...

example 2

Activity of HIF-1α and HIF-1α / VP16 Chimeric / hybrid Constructs in vitro

[0122] The HIF-1α / VP16 hybrid constructs were tested for their ability to activate gene expression by cotransfection along with reporter plasmids into either 293 or HeLa cells (FIGS. 3 and 4, respectively). The reporter plasmids used in these experiments contained the luciferase gene under the transcriptional control of either the erythropoietin enhancer / promoter (Blanchard, K. L., et al., Mol. Cell. Biol. 12:5373-85 (1992)) or the VEGF promoter (Levy, A. P. et al., J. Biol. Chem. 270:13333-40 (1995)). (In FIGS. 3-5, pcDNA3 / HIF / VP16.Afl2 and pcDNA3 / HIF / VP16.R1 are referred to as “HIF-4 / VP-16.AflII” and “HIF-4 / VP-16.R1”, respectively.)

[0123] Prior to transfection, cells were plated in 6 cm dishes at a density of 1×106 (239) or 2.5×105 (HeLa) cells per dish. Cells were co-transfected with the HIF-1α or HIF-1α / VP16 expression plasmids and a luciferase reporter plasmid containing either the EPO enhancer / promoter or ...

example 3

VEGF Production in Response to the HIF-1α / VP16 Hybrid Constructs

[0126] To determine if the HIF-1α / VP16 hybrids were capable of activating expression of an endogenous VEGF gene, these constructs were transfected into HeLa cells and VEGF was assayed by ELISA. Prior to transfection, HeLa cells were plated in 6 cm dishes at a density of 2.5×105 cells per dish. The cells were transfected with 10 μg of the HIF-1α / VP16 hybrid plasmid DNA. At 24 hours post-transfection, cells in duplicate plates were either control (not-induced) or induced by adding fresh media containing 100 μg / ml desferrioxamine. At 40 hours post-induction, the media was harvested and assayed for VEGF by ELISA, (Quantikine™ R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions.

[0127] The results from this assay (FIG. 5) show that the hybrid HIF-1α / VP16 / AflII construct (truncated at amino acid 390 of HIF-1α) was able to activate expression of VEGF even in the absence of induction. The concentratio...

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Abstract

The present invention provides recombinant nucleic acid molecules encoding a chimeric transactivator protein including a DNA binding domain of a DNA binding protein and a protein domain capable of transcriptional activation. 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 transactivator proteins in mammalian cells. Also provided are host cell lines and non-human transgenic animals capable of expressing biologically active chimeric transactivator proteins. In another aspect, compositions and methods for treating or preventing ischemic damage associated with hypoxia-related disorders are provided.

Description

[0001] This application claims priority to PCT patent application PCT / US98 / 25753, filed Dec. 4, 1998, which claims priority to U.S. Ser. No. 09 / 133,612, filed Aug. 13, 1998, which claims priority to provisional application 60 / 067,546, filed Dec. 4, 1997.BACKGROUND OF THE INVENTION [0002] 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. [0003] 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 do...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C07H21/04A61K31/711C12N15/09A61K35/76A61K38/00A61P9/10C07K14/47C07K14/52C07K19/00C12N5/10C12N15/12C12Q1/02
CPCA61K38/00A61K48/00C07K14/4702C07K14/52C07K2319/00C07K2319/71C07K2319/80C12N15/85C12N2799/022A61P43/00A61P9/10
Inventor GREGORY, RICHARDVINCENT, KAREN
Owner GENZYME CORP
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