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Synthesis of a Mini‐Reporter Construct to Test Gene Transfer of RNA Therapeutics.
Author(s) -
Muralidharan Koushik,
Khan Arbaz M,
Fuoco Kerriane,
Patel Hemangi,
Hicks Martin J
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.934.27
Subject(s) - rna splicing , cancer research , alternative splicing , vascular endothelial growth factor , biology , rna , microbiology and biotechnology , gene , exon , vegf receptors , genetics
Glioblastoma multiforme (GBM), a grade IV tumor of the central nervous system, is the most common malignant primary brain tumor in adults, with a median survival of only 14 months. This poor survival rate is due to a lack of efficacy in current therapies, including chemotherapy and radiation, which are limited by the blood‐brain barrier. GBM survival depends on tumor cells to generate new vasculature, which is essential for the exchange of wastes and nutrients. Endothelial cells connect with each other and form the walls of new blood vessels, bridging the gap between the growing tumor mass and the established vasculature of the circulatory system. The membrane receptor that activates this growth is vascular endothelial growth factor receptor 2 (VEGFR2). In our lab, we are developing a novel therapy to alter the VEGFR2 receptor so that it cannot form a bridge with its cognate ligand, vascular endothelial growth factor. Alteration in VEGFR2 so that it cannot form this bridge, blocks its activation and inhibits the development of new blood vessels. We have designed therapies to deliver the genetic sequences of anti‐sense RNA therapy molecules to alter the splicing pattern and expression of the VEGFR2 transcript, creating a soluble VEGFR2 decoy. We have designed and cloned a mini‐reporter‐system that contains the regulatory elements of VEGFR2 splicing. This system measures the efficacy of RNA anti‐sense therapeutics to alter the splicing of the VEGFR2 transcript. The visual marker, eukaryotic green fluorescent protein is used to mimic the natural splicing product, whereas the red fluorescent protein, mCherry detects changes in the the efficacy of our RNA anti‐sense therapy. We use a GBM tissue culture model to measure mini‐reporter and therapy expression. In this manner, the mini‐reporter construct provides a quick and visually measurable test to optimize RNA anti‐sense therapies directed against VEGFR2. Support or Funding Information Bristol‐Myers Squibb