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STUDY OF DENDRITIC CELL TARGETING BY ENGINEERED LENTIVECTORS
by
April M Tai
________________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)
May 2012
Copyright 2012 April M Tai
Object Description
| Title | Study of dendritic cell targeting by engineered lentivectors |
| Author | Tai, April M. |
| Author email | april.tai@gmail.com;april.tai@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemical Engineering |
| School | Viterbi School of Engineering |
| Date defended/completed | 2011-12-16 |
| Date submitted | 2012-05-07 |
| Date approved | 2012-05-07 |
| Restricted until | 2012-05-07 |
| Date published | 2012-05-07 |
| Advisor (committee chair) | Wang, Pin |
| Advisor (committee member) |
Hsiai, Tzung Shing, Katherine |
| Abstract | Dendritic cell (DC) vaccines have great potential as an emerging form of immunotherapy, as DCs are potent antigen-presenting cells, capable of triggering T cell and B cell responses. Our lab has previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. ❧ We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that increasing the amount of high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit α1,2-mannosidase I, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV produced with DMJ (FUGW/SVGmu + DMJ) on 293T.DCSIGN, a human cell line expressing the human DC-SIGN antibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. ❧ In our next study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector through confocal microscopy of GFP-Vpr-tagged virus, drug treatments, and dominant-negative mutants of GTPases, which are necessary for endosomal functions. Using these tests, we demonstrated that our engineered lentiviral vector enters the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis, and that microtubule networks were also involved in a productive infection. Fusion was low-pH-dependent and occurred in the early endosomal stage of transport. Autophagy was also examined for its effect on transduction efficiency. We observed that enhanced autophage activity reduced viral infectivity, while suppressed autophagy boosted transduction efficiency. This study gives us insight on the internalization and trafficking mechanisms used by our engineered vector and gives us tools to improve the efficiency of this platform. ❧ In our last study, we examined the ability of lentiviruses enveloped with an alphaviral envelope glycoprotein derived from Ross River virus (RRV) to mediate transduction of DCs. We found that RRV was only able to specifically mediate transduction of cells through DC-SIGN when the viral vectors were produced under conditions limiting glycosylation to high-mannose glycans. This suggests that these RRV-pseudotyped LVs can be used for DC-targeting, but would require specific conditions during vector propagation for effective targeting infections. |
| Keyword | dendritic cells; lentivector; targeted gene therapy; DC-SIGN |
| 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 |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Tai, April M. |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume4/etd-TaiAprilM-799.pdf |
Description
| Title | Page 1 |
| Full text | STUDY OF DENDRITIC CELL TARGETING BY ENGINEERED LENTIVECTORS by April M Tai ________________________________________________________________________ A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMICAL ENGINEERING) May 2012 Copyright 2012 April M Tai |
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