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GENE DELIVERY TO PULMONARY MUCOSA
by
Jens Erik Harboe-Schmidt
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2006
Copyright 2006 Jens Erik Harboe-Schmidt
Object Description
| Title | Gene delivery to pulmonary mucosa |
| Author | Harboe-Schmidt, Jens Erik |
| Author email | harboesc@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Biochemistry & Molecular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2006-07-06 |
| Date submitted | 2006 |
| Restricted until | Unrestricted |
| Date published | 2006-11-09 |
| Advisor (committee chair) | Kedes, Larry |
| Advisor (committee member) |
Frenkel, Baruch Kasahara, Nori |
| Abstract | We investigated the use of a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with VSV.G for GFP gene transfer to primary rat alveolar epithelial cells (AEC's). Cells grown on plastic exhibited a dose response with transduction efficiencies of 99% at an MOI's of 50. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent and polarized monolayers of AEC's cultured on semi-permeable supports revealed efficient transduction only when lentivirus was applied apically. Manipulating the tight junctions of the monolayers did not enhance infection, indicating that transduction occurred primarily at the apical membrane. In contrast, differentiated tracheal epithelial cells could only be transduced apically in the presence of EGTA but even then at a much lower overall efficiency (15-fold) than was observed for AEC's suggesting that receptor localization may differ between upper and lower airways.; To determine if these vectors could deliver a functional transgene we set out to study sodium pump activity after gene transfer of the alpha1- or beta1 sodium pump subunits of rat Na+,K+-ATPase. Transduction with Lenti-beta1-EGFP was accompanied by coordinate up-regulation of endogenous alpha1 expression, whereas endogenous beta1 expression was unchanged after transduction with Lenti-alpha1-EGFP. Consistent with these findings, transduction with Lenti-beta1-EGFP led to increases in Na+,K+-ATPase holoenzyme as determined by augmentation of sodium pump activity, and therefore supports the feasibility of lentivirus-mediated gene transfer to augment alveolar fluid clearance.; In vivo studies of lentiviral gene delivery to the distal airways in 4 week old rats by non-invasive, intra-tracheal methods led to transduction efficiencies of up to 21% at 2x108 IU. Gene expression in vivo declined more than 5 fold between day 4 and day 25, and was reduced to background levels at day 50. Delivery of 5x107 IU led to infection of 19.6% macrophages, 3.4% AT1 cells and 3.5% AT2 cells suggesting that macrophages were more susceptible to viral infection. However, pulmonary macrophages represent a minor population of airway cells. We furthermore transduced rats intra-tracheally with lentivirus expressing mIL-4 and were able to detect expression in blood plasma (440 pg mIL-4/ml) for up to 28 days before levels were no longer significant. |
| Keyword | gene; delivery; lentivirus; pulmonary; mucosa |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m127 |
| Rights | Harboe-Schmidt, Jens Erik |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-HarboeSchmidt-20061109 |
| Archival file | uscthesesreloadpub_Volume23/etd-HarboeSchmidt-20061109.pdf |
Description
| Title | Page 1 |
| Full text | GENE DELIVERY TO PULMONARY MUCOSA by Jens Erik Harboe-Schmidt A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY AND MOLECULAR BIOLOGY) December 2006 Copyright 2006 Jens Erik Harboe-Schmidt |
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