Abstract
Adeno-associated virus (AAV) gene therapies typically use constitutive transgene expression vectors that cannot be altered after vector administration. Here, we describe a bioorthogonal platform for tuning AAV expression which enables the controlled activation of viral transgenes after transduction. This platform uses a small, synthetic DNA-binding protein embedded in the AAV genome coupled with a heterobifunctional small molecule that recruits endogenous transcriptional machinery to chemically induce transgene expression in a dose-dependent and reversible manner. In human cells, this strategy successfully activates AAV expression across different viral serotypes, cassette configurations, and transgene payloads. Epigenomic analysis reveals that this technology facilitates direct and specific recruitment of the transcriptional regulator BRD4 to AAV genomes. Our results demonstrate that the expression of native AAV genomes can be tuned through chemically induced proximity, opening the possibility of a new class of AAV vectors that can be dynamically potentiated.