Scaled identification of receptor-targeted AAVs for potent and cell type-specific transgene delivery

This interdisciplinary established team initiative will provide the neuroscience community with a toolkit of engineered adeno-associated virus (AAV) reagents for cell type-specific transgene expression in non-transgenic rodents and non-human primates (NHP). While existing AAV capsids engineered for enhanced BBB crossing in macaque NHP from us and others vastly outperform AAV9 after intravenous injection, they remain far short of the potency of equivalent tools in rodent. To achieve functional parity, we will leverage our established track-record in AAV capsid engineering paired with mechanistic insight based on blood-brain barrier (BBB) receptor-mediated transcytosis pathways we recently discovered. The resulting NHP capsids will enable brain-wide payload delivery, e.g. for efficient primate cell type-specific enhancer screening, and utilization of existing gene regulatory element strategies for neuronal cell type specificity (Aim 1). Our approach will utilize structure-informed capsid libraries selected in vitro against BBB receptors known to promote strong transcytosis and we will scale up our screening technologies for discovery of additional novel BBB receptors engaged by AAVs identified through mechanism-agnostic directed evolution. We will generate a toolkit of >60 AAV capsids each for direct and systemic delivery, in addition to >30 AAV cargo miRNA target site designs with distinct neuronal cell subtype-specific expression patterns in mouse (Aim 2). Using ‘null’ AAV capsids that require an additional non- natural membrane protein interaction in order to infect cells, we will mine recently announced comprehensive mouse brain cell transcriptomic atlases and employ a custom automation platform for high throughput in vitro AAV engineering against membrane proteins with cell subtype-specific patterns of expression. In parallel, we will use spatial transcriptomics and machine learning to identify and characterize novel post-transcriptional gene regulatory elements. These capsids and miRNA target sites will synergize with and complement enhancer-AAV strategies for neuronal cell type specificity. To ease adoption of these tools, we will establish a rigorous reagent characterization and dissemination pipeline (Aim 3). Identified reagents will be validated in vivo via quantification of cell type potency and specificity with all metadata and data made available through online repositories. Reagents will be adapted for broad dissemination in plasmid and vector formats via an established collaborative network. Project Number: 1U01MH139785-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Mental Health (NIMH) | Principal Investigator: Viviana Gradinaru (+2 co-PIs) | Institution: CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA, CA | Award Amount: $2,935,208 | Activity Code: U01 | Study Section: Special Emphasis Panel[ZMH1 ERB-E (01)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11118379