Advances in gene transfer methods have created the opportunity for development of gene therapy for human neurological disease such as Parkinsons disease (PD). One goal is to slow dopaminergic neuron loss and the success of such neuroprotective gene therapy is contingent on the development of safe and effective gene transfer vectors that can express a therapeutic gene over long periods of time in specific neuronal populations. The plasmid based herpes simplex virus (HSV) """"""""amplicon"""""""" vectors accommodate a large (9kb) typrosine hydroxylase (TH) promoter and provide highly selective and relatively long term (10 weeks) gene expression in dopamine (DA) neurons in the rat substantia nigra. But, HSV amplicon vector stocks also contain replication defective HSV helper virus which under some conditions and particularly at high multiplicities of infection produces cellular injury and death. The overall goals of this application are three fold:
Aim 1 will compare several complementary new methods to reduce and perhaps eliminate helper virus related toxicity and determine whether they provide increased efficiency and stability of amplicon transgene gene expression in vitro (in cultured primary neurons and astrocytes measuring titers of amplicon and helper, number of lac positive cells and cytotoxicity) and Aim 2 will test their efficacy in vivo using the long tyrosine hydroxylase promotor - b lacZ reporter amplicon (delivering virus to the striatum and scoring bgal expression (mRNA and protein) in DA neurons (TH positive) in the striatum and SN at 1,6, and 16 weeks after infection at several MOIs.). Finally Aim 3 will determine whether the approach of decreasing cytotoxicity leads to a more effective gene therapy. The applicant will construct new amplicon vectors carrying three candidate neuroprotective genes thought to work by different pathways, GDNF, BDNF and bcl-2, use the least cytotoxic system to produce infectious particles, and evaluate them in FluoroGold/6-OHDA lesioned rats, a progressive injury model of PD, examining gene expression characteristics (RNAse protection to measure vector mRNA levels, ELISA and immunostaining to measure gene products), neuroprotection (quantitation of FluoroGold labeled neurons) and neurochemical changes (levels of DA and metabolites-DOPAC, HVA as a measure of both intra and inter-neuron DA turnover and effects on serotonergic neurons with 5-HT, 5-HIAA) at 1 and 4 months after injection of viral stock.
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