Abstract AAV gene therapy has applications for the treatment of diverse genetic diseases and has already been applied in over 1,000 humans to date. Optimistic clinical data using AAV gene therapy has been observed for disorders of the muscle, blood, brain, and those affecting vision. In all these applications, the AAV vectors administered to humans have one thing in common: they are uncontrollable at the level of transgene expression. Following transduction, AAV vector genomes form circular concatemers and limited studies have demonstrated these episomes associate with histones as well as transcriptional activators and repressors. These observations suggest a formal possibility that AAV episomes are, in part, restricted for transgene expression, alluding to the ability to modulate their epigenetic composition to enhance and/or repress the transcriptional activity. We recently developed a technology termed chemical epigenetic modifiers (CEM)s that couple a protein targeting system at a promoter to control gene expression with a specific small molecule activator. This exciting technology has demonstrated specific gene activation and repression at the chromosomal level. In preliminary studies, our technology was evaluated for the recruitment of specific transcriptional and epigenetic regulators to transduced AAV vector episomes. The resultant data demonstrate the involvement of specific epigenetic modifiers to enhance transgene expression greater than 10-fold at a fixed vector dose. Additionally, the unique CEM design allows binding of competitive inhibitors of the activation molecules, thereby providing a mechanism for transcriptional repression. Excited by these findings, we propose a detailed mechanistic examination of this platform for controlled AAV transgene expression in vitro using chromatin immunoprecipitation following cell transduction (Aim 1). Additionally, the functionality of this CEM approach for episomal transgene regulation will be evaluated following systemic AAV administration in a mouse model (Aim 2). Collectively, data generated herein will characterize the natural restriction on AAV transgene expression while investigating a novel approach in vivo for the controlled and specific activation and repression of AAV transgenes towards safer gene therapy applications in general.