The goal of this project is to increase the ability of virus vectors to deliver genes to the postnatal nervous system and to use these vectors to alter cell fate in the nervous system. Two strategies will be undertaken to expand the range and longevity of gene delivery to neural cells in the mouse nervous system. In the first scheme, retrovirus vectors will be generated which can confer stable gene expression on astrocytic cells grafted into the brain. The phosphoglycerate kinase promoter will be used to regulate expression of brain-derived neurotrophic factor (BDNF) for degenerating dopaminergic neurons in newborn weaver mice. These vectors will also be used to genetically modify astrocytic lines capable of migrating in the brain. In addition, one of the astrocytic lines will be converted to a packaging cell line which can be used to deliver retrovirus vectors to endogenous neural cells in vivo. Retrovirus vectors will also be generated to aid in functional analysis of the NF2 gene product in Schwann cells and tumor cells. Further we will try to develop an animal model for neurofibroma formation by retrovirus-mediated delivery of antisense RNA to repress expression of neurofibromin or by homologous recombination to knock-out the normal NF1 allele in Schwann cells from heterozygous NF1 knock-out mice. The behavior of genetically altered Schwann cells will be evaluated in a peripheral nerve injury-repair model. In the second gene delivery scheme we will try to improve herpes vectors for delivery of genes to neurons. A """"""""piggy back"""""""" system of herpes simplex virus vectors will be developed using a combination of amplicon and recombinant virus vectors to combine complementary features of both vector types and to make them mutually dependent on each other. This system will be designed to allow limited replication of the vectors in glia-derived cells using glia-specific promoters to regulate expression of critical genes. Amplicon vectors will be used to deliver NGF and Oct2 to neurons at the time of infection in order to encourage entrance of the virus into latency, at the same time making preparation of vector stocks feasible by using a tet operon system to control expression of these genes in culture. The recombinant virus vector will be deleted for genes contributing to neurovirulence including ICP4 (or ICP27), ribonucleotide reductase, gamma 34.5 and UL41. The herpes vector system will be evaluated for gene delivery, stability of transgene expression and pathogenicity to neural cells in the brain. These new gene delivery systems will provide a basis for therapeutic intervention in animal models of Parkinson's disease and neurofibromatosis.
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