Technical and biological sources of noise confound multiplexed enhancer AAV screening

Abstract Cis-acting regulatory enhancer elements are powerful tools for achieving cell type-specific genetic access in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer discovery remains the accurate characterization of their in vivo expression patterns, which currently relies on labor-intensive, gold-standard one-by-one validation. Here, we evaluate multiple barcoded, multiplexed strategies for accelerated profiling of enhancer-driven expression at cell type resolution. As a proof-of-concept, we test small pools of well-validated enhancer AAVs with known activity across diverse cell types in the mouse brain. Despite extensive optimization and testing, we encounter substantial technical and biological noise, including chimeric AAV packaging products, that obscure true enhancer expression patterns. These effects are particularly pronounced for weaker enhancers and enhancers active in less abundant cell subpopulations. These findings highlight the challenges inherent to multiplexed enhancer AAV screening, the importance of careful enhancer pool design, and the complexity of enhancer AAV biology in vivo.