Optimization of microRNA Target Sequence Arrangement in AAV Vectors Dramatically Enhances Specificity and Efficiency of Transgene Expression in Cortical Microglia

Abstract Microglia play a critical role in diseases such as Alzheimer’s and stroke, making them a significant target for therapeutic intervention. However, due to their immune functions in detecting and combating viral invasion, efficient gene delivery to microglia remains challenging. We achieved specific and efficient gene delivery to microglia using an adeno- associated virus (AAV) vector designed for this purpose. This microglia-targeting AAV vector includes the mouse microglia/macrophage-specific ionized calcium-binding adaptor molecule 1 (mIba1) promoter, green fluorescent protein (GFP), microRNA target sequences (miR.Ts), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and polyadenylation (polyA) signal, positioned between inverted terminal repeats. When miR.Ts were placed downstream of WPRE (between WPRE and polyA), gene expression occurred not only in microglia but also in a substantial number of neurons. However, when miR.Ts were positioned upstream of WPRE (between GFP and WPRE) or on both sides of WPRE, neuronal expression was significantly suppressed, resulting in selective GFP expression in microglia. Notably, positioning miR.Ts on both sides of WPRE achieved over 90% specificity and more than 60% efficiency in transgene expression in microglia three weeks after viral administration. This vector also enabled GCaMP expression in microglia, facilitating real-time monitoring of calcium dynamics and microglial process activity in the cortex. Additionally, intravenous administration of this vector with the blood-brain barrier-penetrant AAV-9P31 capsid variant resulted in extensive GFP expression selectively in microglia throughout the brain. These findings establish this AAV vector system as a robust tool for long-term, specific, and efficient gene expression in microglia.