Our long-term goal is to develop positron emission tomographic (PET) imaging methods that will enable measurement of gene therapeutic efficacy in regenerating dopaminergic terminals. The ability to noninvasively monitor the expression of transduced genes is extremely important for planning treatment of neurodegenerative diseases. Significance of this methodology is very high in developing therapeutic strategies for Parkinson's disease (PD). We propose to use two approaches in order to develop a """"""""marker gene"""""""" for use in the gene activation imaging assay (GALA). The first approach would use an available receptor-radioligand system, which does not, or only minimally, localizes in the regions of interest. For this purpose, we have identified the serotonin 5HTla receptor gene. The second approach employs a gene preferentially expressed in the periphery and not in the CNS. For this, we have identified the serotonin 5HT2b receptor gone. This wild-type gent and its mutant reccptor (i.e. functionality removed) will be developed as a GAIA tool. We propose to use mF-MPPF as a PET probe for the measuring expression of 5HT la receptor. For the 5HT2b receptor we propose to develop imaging probes based on the reported 5HT2b-selective indolyl-urea and pyrimidine class of compounds. In vitro and ex vivo radioligand imaging (autoradiogmphic) studies of rats following intrastriatal adenoviral and lentiviral marker gene delivery will demonstrate validity of this approach. These imaging studies will be accompanied by simultaneous in vitro measurement of mRNA levels and receptor levels at the site of delivery and correlated with imaging probe concentration. This will allow correlation of the concentration of mRNA, receptors and the imaging probe. These methods wilt then be applied to unilaterally lesioned (6-OHDA) rats using """"""""marker gene and GDNF gene"""""""". These rats will also be evaluated by using an assay to measure functionality of the therapeutic GDNF gone and its relationship to the GAlA system by measuring presynaptic dopamine turnover.
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