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ENGINEERING VIRAL VECTORS FOR GENE AND CELL TARGETING
by
Yuning Lei
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 2011
Copyright 2011 Yuning Lei
Object Description
| Title | Engineering viral vectors for gene and cell targeting |
| Author | Lei, Yuning |
| Author email | yuningle@usc.edu; yuning2000@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemical Engineering |
| School | Viterbi School of Engineering |
| Date defended/completed | 2010-11-18 |
| Date submitted | 2011 |
| Restricted until | Unrestricted |
| Date published | 2011-01-25 |
| Advisor (committee chair) | Wang, Pin |
| Advisor (committee member) |
Shing, Katherine Arnold, Donald |
| Abstract | Gene therapy is the introduction of functional genes into dysfunctional cells to treat potentially incurable diseases. The technique sounds promising, yet there are many hurdles must be overcome to make it a practical mean of medicine. Viral vector mediated gene therapy remains one of the most promising gene therapy techniques as its efficiency and duration is the highest among the different gene delivery vehicles. To further refine the viral vector to enhance the gene delivery ability, we designed a strategy by decoupling 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. The underlining mechanism of targeted transduction is that the viral vector will bind to the desirable cell types via the cognate antibody antigen interaction and further be endocytosised into the endosome. Inside the endosome compartment, the low pH environment will trigger the conformation change of the fusogenic molecule which will fuse both the viral and cellular membrane, resulting in releasing the viral core. To further enhance the targeted transduction, we focused on engineering the two parameters on the viral vector. In chapter 2, we engineered the fusion loop of the fusogenic molecule to enhance the activity of the protein. We demonstrated that the engineered fusogenic molecules can enhance targeted transduction many folds. We further looked at the other parameter, the targeting antibody in chapter 3. By designing a single chain antibody, we were able to constructed our targeting virus a simplifier viral production protocol and showed that the viral vector still exhibit targeted transduction. These two chapters concluded our effort in enhancing the targeted transduction by genetically engineering the component displayed on the viral membrane.; The fourth chapter in this report combines the technology of the zinc finger nuclease and the baculoviral vector. Currently, one of the drawback of utilizing integrating viral vector is the possibility of turning on oncogenes when the viral vectors integrated to undesirable sites. In this chapter, we utilize zinc finger nuclease to generate double stranded break and further demonstrate the ability of utilizing this technique to converting the baculoviral vector into gene targeting vector. We showed that our system can targeted transduce both immortalized cell lines as well as human embryonic stem cells. Techniques that we developed here can further enhance the safety of viral gene therapy while retain the efficiency and duration of the viral vectors. |
| Keyword | lentiviral vector; baculoviral vector; CD20 targeting; cell targeting; zinc finger nuclease; gene targeting; human embryonic stem cells; genetic modification |
| 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-m3627 |
| Rights | Lei, Yuning |
| 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-Lei-4245 |
| Archival file | uscthesesreloadpub_Volume26/etd-Lei-4245.pdf |
Description
| Title | Page 1 |
| Full text | ENGINEERING VIRAL VECTORS FOR GENE AND CELL TARGETING by Yuning Lei 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 2011 Copyright 2011 Yuning Lei |
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