StitchR-based dual-AAV delivery strategies enable efficient transduction of defined neural cell populations

Abstract Dual adeno-associated virus (AAV) vector reconstitution has become an indispensable technique for delivering large genes in neuroscience and gene therapy. A novel technology termed StitchR addresses the challenge of low efficiency in dual-AAV reconstruction, yet related tools enabling targeted gene delivery to specific neural cell populations remain insufficiently developed. Herein, we developed four StitchR-based dual-viral reconstitution strategies by combining high-efficiency AAV serotypes with neuron- or glia-specific promoters. Specifically, the AAV11-hSyn-driven StitchR strategy achieved specific transduction of local neurons and their upstream projection neurons (≥ 94.44% specificity); the AAV11-GfaABC1D-driven strategy realized astrocyte-specific transduction (approximately 98.25% specificity); and the AAV11-mIBA1-driven strategy enabled microglia-specific transduction (100% specificity). Furthermore, by incorporating the blood–brain barrier (BBB)-penetrating AAVhu.32-PLUS into the hSyn-driven system, we established a modified strategy that allows brain-wide neuronal transduction. Finally, we confirmed that the StitchR-based dual-viral reconstitution strategy can achieve efficient functional reconstitution of large transgenes in neural cells. Collectively, these dual-AAV delivery strategies expand the valuable AAV toolbox and offer crucial technical support for targeted delivery of large transgenes to defined neural cell populations, holding great significance for basic neuroscience research and potential therapeutic applications.