AAV-DJ enables efficient, function-preserving neuronal transduction for circuit interrogation in human cortical and...

Neural organoids provide tractable models of human brain development, function, and disease, but adeno-associated viral vector (AAV)-based gene delivery is limited by inefficient transduction and insufficient comparative evaluation of the natural and engineered capsids. Here, we evaluated seven AAV capsids (AAV-2, -6, -9, -DJ, -2-retro, -PHP.eB, and -Cap-Mac) for transduction efficiency, cell type tropism, cytotoxicity, and functional integrity in human cortical and thalamic organoids. AAV-Cap-Mac demonstrated the highest transduction efficiency, with broad cellular tropism, but caused cytotoxicity and decreased neuronal activity. By contrast, AAV-DJ yielded efficient neuronal transduction, with preserved calcium dynamics and minimal cytotoxicity, which enabled rabies virus-based circuit tracing and all-optical physiology. Our findings reveal a capsid-dependent trade-off between transduction efficiency and functional integrity and identify AAV-DJ as an optimal serotype for robust gene delivery and physiological interrogation in organoid-based neuroscience. Combining AAV-DJ with emerging molecular and circuit-level technologies in organoids should facilitate mechanistic and translational studies of the human brain.