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ENGINEERING LENTIVIRAL VECTORS FOR GENE THERAPY AND DC-VACCINE
by
Chi-Lin Lee
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 Chi-Lin Lee
Object Description
| Title | Engineering lentiviral vectors for gene therapy and DC-vaccine |
| Author | Lee, Chi-Lin |
| Author email | chilinle@usc.edu;stevenlee.cl@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemical Engineering |
| School | Viterbi School of Engineering |
| Date defended/completed | 2012-02-09 |
| Date submitted | 2012-04-09 |
| Date approved | 2012-04-11 |
| Restricted until | 2012-04-11 |
| Date published | 2012-04-11 |
| Advisor (committee chair) | Wang, Pin |
| Advisor (committee member) |
Shing, Katherine Arnold, Don B. |
| Abstract | An important concept of gene therapy is the delivery of genetic materials to target cells for therapeutic benefit. One of the most important and efficient methods for gene delivery is the use of viral vectors as transfer vehicles. Lentiviral vectors (LVs) derived from human immunodeficiency virus type 1 (HIV-1) are promising vehicles for gene delivery because they not only efficiently transduce both dividing and non-dividing cells, but also maintain long-term transgene expression. In order to enhance the gene delivery ability of the viral vector, we designed a strategy by separating binding and fusion ability of envelope protein into two distinct proteins. By pseudotyping the viral vectors with both an antibody and a fusogenic molecule, we can target and transduce specific cell types. Based on this work, we developed a method to create LVs co-enveloped with the HIV-1 cellular receptor CD4 and a fusogenic protein derived from the Sindbis virus glycoprotein and tested its efficiency to selectively deliver genes into cells expressing HIV-1 envelope proteins. In chapter 2, we demonstrated that this engineered LV can preferentially deliver transgene to HIV-1 envelope-expressing cells in vitro. We conclude that this target LVs give a potential alternative treatment for eradicating HIV-1-infected cells that produce drug-resistant viruses after highly active antiretroviral therapy (HAART). In order to improve the LV transduction efficiency, we introduced the mutations in the E1 domain of Sindbis virus glycoprotein at residues 75 and 237 individually or in combination. The mutation at residues 75 from a neutral and non-polar glycine (Gly, G) to a polar and acidic aspartic acid (Asp, D) can enhance the transduction efficiency by broadening the range of the pH threshold for fusion. In chapter 3 we demonstrated our effort in enhancing the targeted transduction by genetically engineering the fusion component displayed on the viral membrane. To further test lentiviral vectors in preclinical or clinical studies, we constructed a stable producer line for synthesizing DC-SIGN-targeted LVs by a concatemeric array transfection technique could routinely produce vector supernatants with titers above 107 transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. Based on our studies, this production method can generate DC-LVs for preclinical and clinical testing of novel DC-based immunization. |
| Keyword | self-inactivating lentivirus; human immunodeficiency virus; targeted gene delivery; producer cell; inducible gene expression; T cell vaccine; tetracycline |
| 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 | Lee, Chi-Lin |
| 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_Volume3/etd-LeeChiLin-587.pdf |
Description
| Title | Page 1 |
| Full text | ENGINEERING LENTIVIRAL VECTORS FOR GENE THERAPY AND DC-VACCINE by Chi-Lin Lee 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 Chi-Lin Lee |
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