Neuronal death through apoptosis is a central phenomenon in neurological disorders from stroke to neurodegenerative diseases. Thus, understanding the genes and the molecular mechanisms of neuronal death is critical for developing therapeutics for these disorders. Progress in this direction has been slow because of the difficulties in introducing exogenous genes into neuronal cells. Tranzyme, Inc. has developed a novel, safe lentivirus-based system named TranzVector TM, capable of delivering genes with high efficiency to neuronal cells in culture and in vivo. This vector enables novel approaches for investigating gene function and drug target validation in established cell lines with neuronal characteristics. We propose to apply this technology to develop a novel screening system for identifying genes that modulate neuronal apoptosis and to the development of a high throughput reporter assay for drug screening. TranzVector TM will be used to express the well-characterized antiapoptotic genes NGF and Bcl-2 to modulate the function of these genes in normal neuronal PC12 and hNT neurons and those undergoing apoptosis. These studies will validate and optimize the system for the discovery of novel genes that influence apoptosis using gene libraries derived from tissue sources. The system can also be used to evaluate the role of known genes in neuronal apoptosis. A high throughput reporter assay for antiapoptotic agents will also be developed using TranzVector TM containing reporter genes GFP and luciferase linked to the apoptosis-sensitive Pol III promoter of B2-17a RNA. This class of untranslated RNA is induced during apoptosis in cell cultures of PC12 cells and sympathetic neurons and in animal models of stroke. We have chosen a well characterized member of this family, clone B2-17a, for use in the assay. If this promoter shows appropriate regulation in cells undergoing apoptosis, the reporter system can be used in brain slices and whole animals. Further, with the recently demostrated power of the lentiviral vector system in creation of transgenic animals, this reporter system will find wide ranging utility for the production of transgenic animals for the study of apoptosis associated with disease states and developmental processes. These cell and animal models will be applied in PhaseII to the commercial development of protein factors and small molecule drugs to treat neurosensory diseases. Collaboration with other pharma companies will also be established to generate revenue.
