This project will focus on developing novel targeted viral vectors and imaging delivery and transgene expression in tumor cells and in mouse tumor models. Based on over ten years experience developing novel vectors and therapeutic transgenes for tumor therapy, it is clear that one of the major limitations to the effectiveness of this new treatment paradigm is insufficient gene delivery to tumor cells in vivo. In order to compare and optimize different delivery strategies, it is imperative to be able to quantitatively assess vector targeting and transgene delivery in animals, and eventually in human patients. Our goals, then, are to help develop technology, which can be widely applied to a variety of vector/tumor paradigms in order to effect on-site visualization of gene therapy in vivo. Our model vector will be the plasmid-based amplicon vector, which incorporates non-coding elements of herpes simplex virus type 1 (HSV), and hence combines ease of construction, highly efficient gene delivery, and minimal inherent toxicity. These vectors will be targeted to tumor cells by incorporation of the peptide/protein ligands, which bind to tumor- enriched receptors, into the envelope of the virion. These tumor receptors are believed to have an important role in the oncogenesis of specific tumor types and hence serve both as diagnostic markers and eventual therapeutic targets. Virions will be imaged in several ways, including: 1) 111/IN-oxine labeling or genetically modifying the viral envelope protein (gC) with metallothionein known to bind 99m/Tc to image genetically modifying the viral envelope protein (gC) with metallothionein known to bind. 99m/Tc to image mass distribution; and 2) use of proteases as reporters to rapidly monitor transgene expression in cells using previously developed near infrared fluorescent imaging probes (Nature Biotech 1999; 17:375-378). We will attempt to amplify the imaging signals conferred by transgene expression by genetic amplification of the transgene within the host cell nucleus. In addition, we will try to enlarge the domain of imaging by expression of proteins which can be translocated from transduced cells through the extracellular space to surrounding cells or which will mediate fusion of transduced cells with neighboring cells. And finally, we will elicit and monitor tumor cell killing through apoptosis, as mediated by expression of ICE which induces cell suicide. In vivo imaging will play a critical part in this project to test the novel approaches. The developed vector systems may ultimately be used to follow transgene delivery and expression in patients.
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