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Studies of Retroviral-Like-Particles Base Protein Transduction and Antibody-Directed Gene Delivery

by
Dai-Tze Wu
M.S., National Taiwan University, 2007

Thesis Advisors: Monica J. Roth, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Robert Wood Johnson Medical School
Pharmacology Department 4th floor Conference Room
Piscataway

Wednesday, May 7, 2014
1:30 p.m.


Abstract

Gene- and cell-based therapies hold great potential for the advancement of the personalized medicine movement. Gene therapy vectors have made dramatic leaps forward since their inception. Retroviral-based vectors were the first gene transfer vectors to be used in successful gene therapies and still offer the best hope for the long-term correction of many disorders. However, the integration of the shuttle vector within the cellular chromosomal DNA is associated with the risk of insertional mutagenesis, which, in turn, may cause malignant cell transformation. Attempts to overcome these problems, the strategies for using genome-free vectors and re-targeting viral entry by envelope modification have been considered to avoid the unwanted effects of infection and integration. In the studies contained herein we have developed non-infectious MLV virus-like particles (VLPs) that efficiently deliver therapeutic proteins directly into the cytosol and nucleus of living cells in the absence of incorporating viral-encoded genetic material. Two classes of proteins were incorporated into the MLV VLPs: nuclear transcription factors and toxic proteins. Serial exposure to chimeric VLPs allows for continued expression of the transduced protein in the recipient cells, allowing to alteration and regulation of cellular processes without chromosomal integration.

In parallel, we developed antibody-conjugated lentiviral particles that can specifically deliver both visual markers for live-cell imaging as well as selectable markers to stem cell or iPS cells based on the selective expression of stem cell surface markers. The lentiviral vectors pseudotyped with a chimeric Sindbis virus envelope (ZZ SINBIS m168) can conjugate with anti-SSEA4 and anti-CD24 antibodies and mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector also allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. All of these studies have helped to develop safer gene delivery systems for the genetic manipulation of mammalian cells.


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