(Taken directly from the application) Our major objective in this proposal is to design, construct, and test replication-incompetent adenovirus vectors for cystic fibrosis gene therapy. The replication-incompetent vectors should elicit less inflammation after transduction than the replication-defective vectors currently in use, and should therefore increase the efficiency of transduction and the length of time that the transduced gene is expressed. The replication-incompetent vectors will be constructed by deleting adenovirus genes whose products are essential for virus replication and complementing the defective viruses using cell lines that express the virus gene product(s). The effects of E1A, E1B, and E3 expression on the efficiency of transduction and the degree of inflammation will be examined by constructing and comparing viruses that either have these genes present or deleted. The Escherichia coli LacZ gene will be introduced into the viruses as a reporter. The replication-incompetent viruses will then be tested in an inflamed rat lung model of cystic fibrosis to determine the level of inflammation elicited (by staining sections of the transduced lung for inflammatory cells and comparing to untransduced control lung tissue), the efficiency of transduction, the length of expression of the LacZ gene, and the ability of the vector to permit repeated administration without losing transduction efficiency (the latter three tested by staining for beta-galactosidase activity). Using the data generated, the most efficient vector will be designed and constructed. These studies will serve as a prelude to testing of the virus as a gene therapy vector for cystic fibrosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058344-02
Application #
2519628
Study Section
Special Emphasis Panel (SRC (06))
Project Start
1996-09-30
Project End
2000-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Bennett, Michael; Schaack, Jerome (2003) Development of a dual-luciferase fusion gene as a sensitive marker for site-directed DNA repair strategies. J Gene Med 5:723-32
Kohrer, Caroline; Yoo, Jae-Ho; Bennett, Michael et al. (2003) A possible approach to site-specific insertion of two different unnatural amino acids into proteins in mammalian cells via nonsense suppression. Chem Biol 10:1095-102
Zhou, Wenbo; Schaack, Jerome; Zawada, W Michael et al. (2002) Overexpression of human alpha-synuclein causes dopamine neuron death in primary human mesencephalic culture. Brain Res 926:42-50
Galan, Henry L; Bennett, Michael L; Tyson, R Weslie et al. (2002) Inefficient transduction of sheep in utero after intra-amniotic injection of retroviral producer cells. Am J Obstet Gynecol 187:469-74
Cota-Gomez, Adela; Flores, Natalia C; Cruz, Coral et al. (2002) The human immunodeficiency virus-1 Tat protein activates human umbilical vein endothelial cell E-selectin expression via an NF-kappa B-dependent mechanism. J Biol Chem 277:14390-9
Thomas, D L; Schaack, J; Vogel, H et al. (2001) Several E4 region functions influence mammary tumorigenesis by human adenovirus type 9. J Virol 75:557-68
Orlicky, D J; DeGregori, J; Schaack, J (2001) Construction of stable coxsackievirus and adenovirus receptor-expressing 3T3-L1 cells. J Lipid Res 42:910-5
Rich, T C; Tse, T E; Rohan, J G et al. (2001) In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J Gen Physiol 118:63-78
Orlicky, D J; Schaack, J (2001) Adenovirus transduction of 3T3-L1 cells. J Lipid Res 42:460-6
Bennett, M; Galan, H; Owens, G et al. (2001) In utero gene delivery by intraamniotic injection of a retroviral vector producer cell line in a nonhuman primate model. Hum Gene Ther 12:1857-65

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