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- Title
873. Development of High Throughput Functional Screening of Cardiovascular Therapeutic Genes Using the HVJ-E Vector.
- Authors
Nishikawa, Tomoyuki; Nakagami, Hironori; Matsuki, Atsuhi; Saito, Yukihiro; Morishita, Ryuichi; Tamai, Katsuto; Kaneda, Yasufumi
- Abstract
Isolation of effective therapeutic genes to control angiogenesis is critical for the advancement of gene therapy for various diseases, including cardiovascular diseases and cancer. The goal of the present study is to screen a human cDNA library using Hemagglutinating Virus of Japan envelope (HVJ-E) vector to isolate candidate genes to regulate endothelial cell growth with potent therapeutic potential. cDNA library were randomly infused into HVJ-E vector, which were then transferred to Human Aortic Endothelial Cells (HAEC) that were seeded in 96-well plates. Then, the MTS assay revealed variable HAEC proliferation among individual wells. To characterize the candidate genes responsible for inducing or reducing endothelial cell proliferation, DNA was extracted from the cell population showing the highest or lowest degree of proliferation and was then transformed directly into E. coli. Use of this system allowed isolation of candidate genes rapidly, because HVJ-E vector does not require preparation of a viral library or packaging cell constructions. In addition, use of this vector allows easy cloning of candidate genes by transformation of E. Coli (12–16 hours). These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes.We further evaluated the candidate genes by c-fos promoter assay, and finally got three pro-angiogenic and two anti-angiogenic candidate genes. Interestingly, one of the identified growth suppressive gene is also known as CHMP1B or CHMP1.5, a human homologue of yeast SNF7p (sucrose non-fermenting). The CHMP family of proteins participates in intracellular membrane traffic event, of which the endocytic pathway is an important component. Further, a recent study reported that CHMP 1B binds to spastin, a protein that is abnormal in the most common form of pure hereditary spastic paraplegia. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.Molecular Therapy (2006) 13, S336–S336; doi: 10.1016/j.ymthe.2006.08.961
- Subjects
GENE therapy; CANCER; SENDAI virus; CELL proliferation; NEOVASCULARIZATION; ESCHERICHIA coli
- Publication
Molecular Therapy, 2006, Vol 13, pS336
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.961