Isolation of the gene responsible for Cystic Fibrosis (CF) in 1989 suggested new therapies based on somatic gene transfer into epithelial cells of affected organs. The subsequent demonstration of correction of the CF defect in cultured cells by viral mediated transduction of the CF gene (called CFTR) was the first step toward this goal. However, many important questions remain. We describe in this proposal a human xenograft model of CF that may be extremely important in answering these questions. Epithelial cells isolated from airways of normal and CF patients (nasal, trachea, and bronchus) are seeded into denuded rat trachea which are implanted into the flanks of nu/nu mice. Over the ensuing 6 weeks a fully differentiated human epithelium develops in the seeded grafts which is indistinguishable morphologically from normal airway epithelium. We have revised this system in the following way to form a genetically modified epithelium. The isolated epithelial cells are plated in primary culture, exposed to recombinant retroviruses, harvested, and seeded into denuded grafts. We have achieved transgene expression in up to 50% of the fully differentiated epithelium in vivo using lacZ retroviruses. Primary CF epithelial cells exposed to CFTR transducing retroviruses will be used to reconstitute denuded trachea. The resulting epithelium will be analyzed at the light and electron microscopic level to identify any morphological consequences of constitutive and high-level CFTR expression. Electrophysiological analysis of the grafts will address important issues of complementation in this relevant setting. Fully reconstituted trachea will also be used to study and compare strategies for in vivo gene delivery. Recombinant lacZ expressing retroviruses, adenoassociated viruses, and adenoviruses will be directly introduced into the lumen of the reconstituted grafts as they reside subcutaneously in the nu/nu mice. The grafts will subsequently be analyzed for efficiency of gene transfer and stability of gene expression. Virus systems which show the most promise will be used to directly introduce CFTR into the reconstituted grafts in vivo. Grafts transduced with CFTR in vivo will be analyzed using the morphological and functional criteria described above.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL049040-01
Application #
3368149
Study Section
Special Emphasis Panel (SRC (MP))
Project Start
1992-08-01
Project End
1993-03-31
Budget Start
1992-08-01
Budget End
1993-03-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Zhang, Zhe; Reenstra, William; Weiner, Daniel J et al. (2007) The p38 mitogen-activated protein kinase signaling pathway is coupled to Toll-like receptor 5 to mediate gene regulation in response to Pseudomonas aeruginosa infection in human airway epithelial cells. Infect Immun 75:5985-92
Kobinger, Gary P; Limberis, Maria P; Somanathan, Suri et al. (2007) Human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells. Hum Gene Ther 18:413-22
Bell, Peter; Vandenberghe, Luk H; Wu, Di et al. (2007) A comparative analysis of novel fluorescent proteins as reporters for gene transfer studies. J Histochem Cytochem 55:931-9
Zhang, Zhe; Louboutin, Jean-Pierre; Weiner, Daniel J et al. (2005) Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by Toll-like receptor 5. Infect Immun 73:7151-60
Bals, R; Wilson, J M (2003) Cathelicidins--a family of multifunctional antimicrobial peptides. Cell Mol Life Sci 60:711-20
Medina, Maria Fe; Kobinger, Gary P; Rux, John et al. (2003) Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung. Mol Ther 8:777-89
Wang, Xiaorong; Moser, Christian; Louboutin, Jean-Pierre et al. (2002) Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung. J Immunol 168:810-5
Moser, Christian; Weiner, Daniel J; Lysenko, Elena et al. (2002) beta-Defensin 1 contributes to pulmonary innate immunity in mice. Infect Immun 70:3068-72
Croyle, Maria A; Chirmule, Narendra; Zhang, Yi et al. (2002) PEGylation of E1-deleted adenovirus vectors allows significant gene expression on readministration to liver. Hum Gene Ther 13:1887-900
Bals, R; Weiner, D J; Meegalla, R L et al. (2001) Salt-independent abnormality of antimicrobial activity in cystic fibrosis airway surface fluid. Am J Respir Cell Mol Biol 25:21-5

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