The goal of this application is to effectively and selectively eliminate B-cell derived lymphoma and leukemia cells in cancer patients using a naturally targeted viral vector system. To achieve this goal, the applicant proposes to develop a novel combinatorial gene therapy approach as applied to Burkett B lymphoma based on i) a minimal """"""""gene-less"""""""" B lymphotropic Epstein-Barr virus (miniEBV) vector, and ii) a genetically enhanced """"""""hyper-suicide"""""""" HSV1 thymidine kinase (super-TK). Specifically, the following studies will be undertaken:
Aim 1) To test the efficacy of B lymphotropic miniEBV vectors to deliver and express a suicide gene into human B-cell lymphomas following oncotropic and oncolytic strategies. This approach is based on the transfer of the viral thymidine kinase (TK) gene into B-cell derived lymphomas rendering them sensitive to the prodrug ganciclovir (GCV). For this endeavor miniEBV/sTK will be used to infect B-lymphoma cells in vitro and the transiently infected cells implanted in an animal model to analyze prodrug mediated eradication of the lymphoma using ex vivo protocol.
Aim 2) Development of an in vitro cultured packaging cell system to produce helper-free infectious miniEBV. This will involve cloning the genome of EBV in a BAC based vector to delete the packaging sequence by homologous recombination. In addition, EBV negative cell lines will be evaluated for their permissivity to miniEBV replication and packaging into infectious virions.
Aim 3) Use the results obtained in Aims 1-2 to test the miniEBV system for its efficiency and safety in a SCID-Human lymphoma/leukemia animal model using an in vivo protocol. Pre-established human B-lymphoma in vivo by intravenous (i.v.) injection with this system will be also evaluated in order to eliminate the disseminated B-lymphoma from various organs of the animal.
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