1 2 3 4 5 pUNISHER : A high level expression cassette for use with recombinant viral vectors for 6 rapid and long term in vivo neuronal expression in the CNS 7 8 9

40 Fast onset and high level neuro-specific transgene expression in vivo is of importance for many 41 areas in neuroscience, from basic to translational, and can significantly reduce the amount of 42 vector load required to maintain transgene expression in vivo. In this study, we tested various cis 43 elements to optimize transgene expression at transcriptional, post-transcriptional and post44 translational levels and combined them together to create the high level neuronal transgene 45 expression cassette, pUNISHER. Using a 2 generation Adenoviral vector system in 46 combination with our high level expression cassette, pUNISHER, we characterized its rate of 47 onset of detectable expression and levels of expression compared to a neuro-specific expression 48 cassette driven by the 470 bp human synapsin promoter in vitro and in vivo. Our results 49 demonstrate in primary neurons that pUNISHER cassette, in a recombinant Adenovirus (rAd) 50 Type 5 background, led to a faster rate of onset of detectable transgene expression and higher 51 level of transgene expression. More importantly, this cassette led to highly correlated neuronal 52 expression in vivo and led to stable transgene expression up to 30 days in the auditory brainstem 53 with no toxicity on the characteristics of synaptic transmission and plasticity at the calyx of Held 54 synapse. Thus, the pUNISHER cassette is an ideal high level neuronal expression cassette for 55 use in vivo for neuroscience applications. 56 57