HSV-1 vectors are attractive for gene therapy of aging disorders that affect the brain because HSV-1 can persist indefinitely in neurons in the latent state and large HSV-1 vectors can coexpress multiple genes. This laboratory has developed a Herpes Simplex Virus (HSV-1) plasmid vector system for gene transfer into neurons. Using this system, we have begun to explore gene therapy approaches to specific aging disorders that affect the brain, such as Parkinson's Disease (PD). We have shown that delivery of a HSV-1 vector that expresses human tyrosine hydroxylase into the partially denervated striatum in the 6-hydroxydopamine rat model of PD results in long-term (1 year) biochemical and behavioral correction. Other investigators have demonstrated the potential of using this vector system for gene therapy for a number of other neurological disorders. We developed a helper virus-free packaging system for these HSV-1 vectors. This improvement substantially reduces the cytopathic effects and the inflammatory response previously associated with gene transfer. Furthermore, we and others have recently identified specific promoters that support long-term expression in rat forebrain neurons. However, the relatively low titers remain one of the primary barriers to the use of this vector system for human gene therapy of aging disorders that affect the brain. The goal of this proposal is to develop a packaging cell line that can produce high-titer helper virus-free HSV-1 vector stocks. High-titer retrovirus, lentivirus, and adenovirus vector stocks have been produced using packaging cell lines, and these vector stocks have been used in human gene therapy. The first specific aim will isolate a cell line that stably maintains an HSV-1 genome that does not express any immediate early (IE) genes and lacks a packaging site. The second specific aim will produce high-titer, helper virus-free HSV-1 vector stocks by using an HSV-1 vector that contains the 3 essential IE genes flanked by lox sites. This packaging system is based upon standard genetic complementation. High-titer vector stocks will be produced by serial passaging. To excise the IE genes from the vector, the final passage will use a cell line that expresses Cre recombinase. The third specific aim will evaluate the safety features of this packaging system. The fourth specific aim will use these high-titer vector stocks to achieve gene transfer to large numbers of cells in the rat striatum.
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