The long-term goal of this project is to develop novel gene therapies for neurodegenerative diseases. In the previous support period, we focused on adenoviral (Ad) vectors to deliver the gene encoding GDNF (glial cell line-derived neurotrophic factor). Ad-GDNF injected into either the substantia nigra or striatum of a progressive degeneration model of Parkinson's disease protected dopaminergic (DA) neurons against cell death induced by the neurotoxin 6-OHDA. Ad-GDNF injected into the striatum also prevented the acquisition of behaviors and molecular changes that occurred in DA deficient young and aged rats. This proposal focuses on the hypothesis that anti-apoptotic gene delivery will also protect DA neurons in vitro and in vivo and have a synergistic effect with delivery of neurotrophic factor genes. Viral vectors harboring genes that block specific apoptotic death pathways, including XIAP, a dominant-negative caspase-9, bcl-2 and bclxl will be studied for effects on survival and function of DA neurons either alone or in combination with neurotrophic factors, GDNF or neurturin. Genes will be delivered to DA neurons in culture and in rat brain using helper free HSV:AAV hybrid amplicon vectors. These vectors will incorporate bidirectional expression cassettes that drive both the therapeutic gene and the cellular marker gene, green fluorescent protein, to permit specific evaluation of transduced cells. Expression will be controlled using the tetracycline responsive element such that transgene expression is """"""""on"""""""" in the presence of tetracycline activator (TA) and in the absence of doxcycline (Dox). Vectors will be made in which TA is driven by a viral promoter of the DA cellular promoter, tyrosine hydroxylase (TH). Effects of the 'therapeutic' genes will be studied using non-neuronal cells, the DA cell line, MN9D, and primary fetal DA neurons treated with the neurotoxins, MPP+ or 6-OHDA or other cellular insults. In vivo effects of therapeutic genes will be studied in: 1) rats that have received grafts of fetal DA neurons, and 2) rats that have received a progressive 6-OHDA lesion of the nigrostriatal projection. Reversibility of effects will be studied by administration of Dox. Effects on DA neurons will be evaluated using quantitative morphometric and molecular techniques and behavioral evaluations. This project also aims to continue its evaluation of new generation viral vectors, including E2b deleted Ad, totally gutted Ad, and HSV:AAV amplicon, for stability and levels of expression in the nigrostriatal system. The studies involve collaborations among investigators at Children's Memorial Hospital and Northwestern Univ. Med. School and are relevant to the development of novel therapies for neurodegenerative diseases and injuries to the CNS.
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