Gene therapy is a highly promising strategy for a wide variety of biomedical applications including cancer treatment, immunization and gene restoration. VP22 possesses novel trafficking ability where protein produced in one expressing cell, traffics to the nuclei of neighboring non-expressing cells. Further, VP22 chimerics can carry large, effector proteins without altering the function of the attached proteins. In addition to trafficking, VP22 has novel intracellular localization properties including microtubule association and nuclear targeting. Although most VP22 data have been obtained from studies with herpes simplex virus (HSV-1), we have found bovine herpesvirus-l (BHV-l) VP22 to have improved biotherapeutic potential compared to HSV-VP22. Importantly, BHV- 1 VP22 can traffic a fused effector protein up to 20 times more efficiently than HSV-l. BHV- and HSV-VP22 possess only 28.7 percent amino acid homology with numerous motif differences suggesting the opportunity for considerable diversity in structure and function. Our long-term goal is to maximize VP22-mediated gene therapy by defining VP22 intercellular transport mechanisms as well as in vivo repercussions regarding VP22 biotherapeutic delivery. To accomplish our long-range goal, we have plasmids expressing VP22 of BHV-1 as well as defined VP22 mutants and will elucidate the mechanisms of VP22 that contribute to gene delivery by achieving the following Objectives: 1. We will functionally map the regions of VP22 that govern trafficking and localization. (a) We will engineer truncations of VP22 to evaluate regions responsible for trafficking and nuclear localization. We will build on our data that that the carboxy-terminal half of VP22 is essential for trafficking. (b) We will assess the effects of VP22 mutants (tyrosine residues that are phosphorylated and two important cysteines in VP22) on trafficking and nuclear localization. (c) We will utilize a novel cross-linking agent and MALDI-mass spectrometry to identify the specific interaction of VP22 with nuclear and cytoplasmic proteins. 2. We will develop and analyze VP22 delivery of thymidine kinase to tumors for suicide gene therapy. (a) We will evaluate the efficiency of VP22 delivery using a suicide gene therapy approach, where fusion genes will be constructed encoding VP22-tk chimeric polypeptides. This construct will be compared to vector-tk only by testing in vitro. (b) We will also evaluate the efficacy of VP22-tk chimeric polypeptides in vivo tumor killing in the presence of ganciclovir.
