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Rtef-1 variants and the use thereof for inhibition of angiogenesis

A technology of RTEF-1 and expression vector, applied in the field of molecular biology

Inactive Publication Date: 2011-02-09
RES DEVMENT FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, current VEGF-blocking treatments typically result in inhibition of the interaction of extracellular VEGF with the corresponding cell surface receptors

Method used

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  • Rtef-1 variants and the use thereof for inhibition of angiogenesis
  • Rtef-1 variants and the use thereof for inhibition of angiogenesis
  • Rtef-1 variants and the use thereof for inhibition of angiogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] experiment method

[0185] Isolation and culture of primary ocular vascular endothelial cells

[0186] All human cells and tissues were used in accordance with approved Institutional Review Board methods. Primary cultures of endothelial cells isolated from human retina were established using established methods and used as a source of mRNA (Kanda et al., 1998; Silverman et al., 2005). Human cadaver eyes were obtained from unidentified donors (Lion's EyeBank, Portland, OR) within 24 hours of death. Donors had no history of cardiovascular or ocular disease and ranged in age from 16-42. Briefly, these retinal and iris tissues were aseptically dissected and isolated from donor eyes and digested in 0.2% collagenase (Sigma Chemical Co, St Louis, MO) Mouse monoclonal antibody-coated magnetic beads (Dynal Biotech, Inc., Lake Success, NY) separate endothelial cells (EC) from other cell types. ECs were cultured in MCDB-131 complete medium (Clonetics / BioWhittaker, Walkersville...

Embodiment 2

[0198] A novel isoform of RTEF-1 is present in hypoxic and normal ocular vascular endothelial cells

[0199] Using the F1 and R1 primer pairs, cDNA amplification from primary cultures of human retinal (PRVEC) and iris (PIVEC) vascular endothelial cells was amplified to obtain approximately 1305bp and 936bp products ( figure 1 B). Using the same primer pair, amplification of cDNA isolated from PRVEC that had been cultured under hypoxic conditions for 24 hours prior to isolation of mRNA yielded an additional product of approximately 447 bp ( figure 1 B). 651bp cDNA was isolated from human primary retinal vascular endothelial cells (PRVEC).

[0200] Sequencing analysis revealed that the largest product was identical to the full-length 1305bp RTEF-1 gene spanning the start and stop codons (SEQ ID NO: 1), while the 936bp, 651bp and 447bp transcripts were alternatively spliced ​​transcripts of the 1305bp product things. The following description of codons will be numbered accord...

Embodiment 3

[0208] Effect of new RTEF-1 isoforms on expression from the VEGF promoter

[0209] The polypeptide from the 1305bp isoform has been shown to act as a transcriptional stimulator of VEGF in bovine aortic endothelial cells by binding to the Sp1 site (Shie et al., 2004). A study was therefore carried out to investigate whether the new isoform was able to stimulate expression from the human VEGF promoter. The 5' proximal promoter of the human VEGF gene containing 54 bp of the 5' UTR and 1,082 bp upstream of the transcription start site was cloned into the pSEAP reporter plasmid and the RTEF-1 isoform into the pcDNA expression vector. Due to difficulties in nucleofection of plasmid DNA into primary cultured ocular vascular endothelial cells, the 293T cell line was used as an alternative cell line for transfection studies. Co-transfection of the VEGF promoter-reporter plasmid with one of the RTEF-1 isoforms showed that the 1305bp, 936bp and 447bp isoforms upregulated the expression ...

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Abstract

Dominant negative (DN) variants of transcriptional enhancer factor 1- related (RTEF-I) are described. DN RTEF-I polypeptides may be directly targeted to cells or delivered in nucleic acid expression vectors to alter cellular transcription. Methods for inhibiting VEGF production and thereby treating angiogenic disorders such as cancer are described. For example, in certain aspects, DN RTEF-I may be used to treat angiogenic disorders of the eye such as age related macular degeneration (AMD).

Description

[0001] This application claims priority to US Application No. 60 / 942,249, filed June 6, 2007, the entire disclosure of which is expressly incorporated herein by reference in its entirety. technical field [0002] The present invention relates to the field of molecular biology and in particular to the process involved in the formation of blood vessels (angiogenesis). Background technique [0003] Transcription enhancer factor 1-related (RTEF-1) genes are members of the TEA DNA-binding domain gene family. The TEA DNA binding domain gene family is highly conserved from Aspergillus nidulans, yeast, Drosophila, mouse to human. TEA DNA-binding family proteins can be involved in the activation or repression of different genes, and these specific functions of them can be altered by binding to other proteins (Kaneko and DePamphilis, 1998). Expression of specific members of these genes has been identified in a variety of mammalian tissues, including heart, skeletal muscle, pancreas, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/16
CPCC07K14/435A61K38/00A61K45/06A61K38/10A61K38/1709A61K38/16A61P13/12A61P27/02A61P29/00A61P35/00A61P35/04A61P9/00C12N15/11C12N15/85C12N15/86A61N5/10C07K7/06C07K14/4703A61K9/0048A61K48/00C07K7/08C07K16/00C07K2319/02C07K2319/10
Inventor J·蒂莫西·斯托特特雷弗·麦可法兰德本诺伊·阿普库坦
Owner RES DEVMENT FOUND
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