Biochemical Investigation of the Intracellular Trafficking of Non-Viral and Hybrid Gene Therapy Vectors
Drake, David Michael
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https://hdl.handle.net/2142/82422
Description
Title
Biochemical Investigation of the Intracellular Trafficking of Non-Viral and Hybrid Gene Therapy Vectors
Author(s)
Drake, David Michael
Issue Date
2009
Doctoral Committee Chair(s)
Daniel Wayne Pack
Department of Study
Chemical Engineering
Discipline
Chemical Engineering
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Cell
Language
eng
Abstract
We also used reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) applied of post-transfection time increments to determine the timeframe of reverse transcription, thereby estimating endosomal escape of VLP. MLV were observed to achieve reverse transcription rapidly, with more than half of infecting viruses being reverse transcribed within 8 hours. PEI:VLP were delayed approximately 4 hours (relative to viruses) in having their RNA reverse transcribed. This suggests VLP experience delayed endosomal escape, unpackaging, or release of the capsid/reverse transcription complex from the lipid bilayer. This delay likely accounts for much of the inefficiency observed in PEI:VLP relative to MLV. (Similar experiments attempted with integrase inhibitors did not yield useful results.) Improvement of hybrid vectors probably depends on expediting the escape of VLP from the endolysosomal network and the VLP lipid bilayer. This might be accomplished by developing polymers with stronger endosomolytic properties and/or by the incorporation of fusogenic peptides to assist in endosome and bilayer escape.
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