Abstract Recombinant adeno-associated virus (rAAV) is a non-replicative DNA viral vector that is used in three FDA approved gene therapies and many ongoing clinical trials. Despite the popularity of rAAV as a therapeutic vehicle, there exists no generalizable method of tightly controlling rAAV transgene dose. After transduction, most rAAV genomes persist as concatemeric episomes that are retained over time in non-proliferating cells. Recent evidence suggests that rAAV episomes are chromatinized and epigenetically regulated, although further inquiry is needed to clarify the relationship between epigenetic modifications and rAAV transgene expression. The Hathaway Laboratory has developed a novel, titratable, and reversible system of epigenetic editing that uses bifunctional small molecules, called Chemical Epigenetic Modifiers (CEMs), to recruit endogenous epigenetic machinery to targeted genetic loci. We hypothesize that potent and specific next-generation CEM compounds (SLF* CEMs) could be applied to chemically regulate rAAV transgene expression. In Aim 1, SLF* CEM-based systems of rAAV regulation will be constructed and screened for efficacy in human cell lines. In Aim 2, the putative epigenetic mechanism of CEM-mediated rAAV regulation will be interrogated through knock down/replacement and CUT&RUN-Sequencing experiments. The results of this study could provide a foundation for the development of a system of epigenetically tuning the dose of rAAV- based gene therapies without the need for vector readministration.