RNA packaging and gene delivery using Tobacco mosaic virus pseudo virions

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Hung, Chi-Wei
Bentley, William
RNA interference (RNAi) has emerged as a powerful tool for the study of gene function and post-transcriptional regulation. However, the lack of a proper delivery system for RNAi is a major problem for its application as a therapeutic agent. In this study, <em>Tobacco mosaic virus</em> (TMV) is utilized as an RNAi carrier for gene delivery into mammalian and insect cells. The self assembly and disassembly of TMV is investigated to create chimeric viruses for gene delivery. The origin of assembly sequence (OAS) within the TMV RNA initiates its association with coat protein through a unique hairpin structure. Studies in this dissertation show that by incorporating TMV OAS into an RNA of interest, the RNA can assemble into "pseudo-virions" by the virus coat protein. The length of the pseudo-virions changed in proportion with the size of the RNA. To deliver the RNA to the targeted cells, virions are further surface-modified with synthetic cell-penetrating peptides to facilitate cell endocytosis. Two genes were selected as targets: 1) EGFP as a visual marker and 2) Cyclin E for control of cell cycle. EGFP is expressed in a transient expression experiment using a plasmid vector, pEFGP-N1. Cyclin E is regulated endogenously in High FiveTM cells, and its translation is targeted using the pseudo virions. Pseudo-virions targeting EGFP RNA (antisense EGFP) are able to suppress transient EGFP production by 61% whereas pseudo virions targeting cyclin E (antisense cycE) are capable of arresting cells at G1 phase. This RNA packaging system protects packaged RNA and provides a means of delivering RNAi constructs into various host cells.