99. Construction and Evaluation of Recombinant AAV Vectors for Central Nervous System Gene Delivery

The Adeno-associated virus (AAV) have become a highly promising tool for research and clinical applications in the Central Nervous System (CNS). Here we used the reporter gene EGFP under the transcriptional control of five minimal promoters (MinP) that show efficient and specific expression in neurons or astrocyte in vitro. We also describe the extent of viral spread, transduction efficiency and cell type specificity of each promoter into the mouse striatum using the AAV serotype 8 (AAV8). Robust and specific neuronal EGFP expression was observed with the BM88 (88pb) and B2RN (170bp) MinPs, both in vivo and in vitro. Cell typing with immunofluorescence confirmed the efficient AAV8 gene expression into the striatal neurons. Furthermore, we detected axonal transport of the EGFP protein when using these promoters. Additionally, two variants of the minimal human GFAP promoter (<600bp) were evaluated to analyze the role of a 75bp segment (D region) spanning bp -132 to -57 with respect to the RNA start site, in the control of the transgene expression. A reduction on the transcriptional activity was observed in vivo when the region was eliminated. In addition, a minimal murine GFAP promoter (581bp) was generated that exhibited mostly glial expression; however, we also observed EGFP expression in other type of cells like microglia. In summary, we have developed a set of AAV vectors designed for SNC specific cell type expression using minimal promoters to drive gene expression when the size of the inserts matters.