900. Specific AAV Serotypes Stably Transduce Hippocampal and Cortical Cultures with High Efficiency and Low Toxicity

Cultures of dissociated neurons and astrocytes are frequently used by neurobiologists to study neuronal physiology and pathophysiology. However, current methods for gene delivery into neurons in vitro are limited by very low efficiency, short-term gene expression, toxicity and/or the requirement to transfect on the day of plating. We hypothesized that adeno-associated viral (AAV) vectors might represent an efficient and less toxic method for neuronal transduction. We tested AAV transduction of primary cultures of 150,000 E18 rat cortical or hippocampal neurons and astrocytes using six serotypes (AAV1, 2, 6, 7, 8, and 9), each containing the same AAV-2-based genome encoding CMV-GFP.Mature cultures of hippocampal neurons were transduced at 4 weeks post-plating by AAV1, 2, 7, 8 and 9, resulting in GFP expression for at least 1 week. AAV2 (2.5 E+11 genome equivalents, GE) was particularly effective in transducing the mature hippocampal cultures. Expression of GFP was detectable by 1 week post-transduction in 40-60% of the cells, localized predominantly in cells expressing the neuronal marker MAP2, and stable for up to 4 weeks (the life of the culture) without inducing cell death. Immature hippocampal cultures were exposed at 1 day post-plating to AAV1, 2, 6, 7 and 9, and transduced by AAV1, 2, 7 and 9. The AAV9 serotype (5E+11GE) transduced 50-70% of cells in the d1 hippocampal cultures and GFP expression was stable for 4 weeks. The AAV6 serotype caused cell death in both the mature and immature hippocampal cultures and the AAV7 serotype transduced both neurons and astrocytes. To determine whether cortical neurons were transduced by the same vectors, AAV1, 6, 7 and 9 were applied to cortical cultures at 4 weeks post-plating. All 4 serotypes transduced cortical cells and the GFP expression was stable for 1-2 weeks. In both hippocampal and cortical cultures, cell death occurred at later time points (2-4 weeks) after transduction although toxicity was not related to the volume of vector applied or GFP expression. Work is ongoing to quantify the percentages of neurons and astrocytes transduced by each serotype and to explore the in vitro transduction patterns of AAV vectors with engineered capsid proteins. These results should make possible a wide range of neurobiological experiments in cultured neurons, including delivery of shRNAs. Supported by NS040978 (DW) and P30-DK-047757.