Gene transfer in the mammalian nervous system has been the primary research focus of our laboratory for the past decade. We are excited that this RFA has come at a time when the field is flourishing, yet clinical translation remains daunting, and much work needs to be done for ultimate success in the clinic. In this grant application we propose to focus on some of the more pressing needs using rat models of Parkinson Disease.
Our first aim i s to further develop more efficient and readily packaged and purified AAV vectors for clinical translation. Here, we will characterize and compare pseudotyped and chimeric AAV vectors and in addition develop novel reagents, including helper plasmids and protocols which can be used by the entire gene therapy community to more efficiently generate these vectors. Our preliminary data suggests that these new chimeric and pseudotyped vectors represent a significant advance above our current generation rAAV-2 vectors. Secondly, we will develop optimal expression cassettes with a focus on promoter; post regulatory sequences as well as elements like the human beta-interferon scaffold attachment region (SAR) to boost expression. Thirdly, we will further develop a regulatable system. We present in our preliminary data our latest generation bi-directional tet cassette with tandem minimal insulator sequences flanking the vector genome. Here we propose to use this vector as the starting point to develop a novel cassette with the use of KRAB-AB domain from kid-1 as a suppressor.
Our fourth aim i s the use of rAAV to over express PAEL receptor in the adult rat substantial nigra with characterization of the phenotype as a potential genetic model of Parkinson Disease. Finally, we propose the use of a picospritzer and in vivo single unit recording to develop methods for focal and electrophysiological mapped neuronal gene delivery. We will target the substantia nigra pars compacta, using AAV expressing wildtype parkin, as a potential therapy for parkin mutation associated, autosomal recessive Parkinson Disease (AR-PD) as modeled by the PAEL receptor over expressing rats as developed in specific aim 4.
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