Spatial mapping of barcoded, brain-tropic AAVs using multiplexed RNA in situ hybridization.

Area of Science:

• Neuroscience
• Molecular Biology
• Biotechnology

Background:

• Gene therapy using adeno-associated virus (AAV) vectors faces challenges in delivering therapeutic cargo to specific central nervous system (CNS) cell types.
• Precise targeting is crucial for developing effective AAV-based therapies for neurological disorders.

Purpose of the Study:

• To utilize high-plex in situ transcriptomics for high-resolution spatial mapping of AAV payloads in the intact mouse brain.
• To profile the tropism of 22 barcoded AAV variants, including novel AAV9 derivatives, to identify vectors with enhanced cell-type specificity in the CNS.

Main Methods:

• Employed 10× Genomics Xenium (high-plex in situ transcriptomics) to spatially map barcoded AAV payloads at subcellular resolution.
• Conducted tropism profiling of 22 barcoded AAV variants, assessing their transduction patterns in various CNS cell types.
• Analyzed AAV biodistribution and blood-brain barrier (BBB) traversal capacities.

Main Results:

• Established AAV vectors (AAV9-PHP.eB, AAV9-CAP-B10) showed distinct neuronal and non-neuronal subtype preferences.
• Several novel variants demonstrated tropism for endothelial and vascular cells, indicating limited BBB penetration.
• Three novel variants (AAV9-BTX166, -BTX168, -BTX175) showed enhanced endothelial/mural cell tropism.
• Two novel variants (AAV9-BTX149, -BTX001) selectively targeted specific inhibitory neuron subtypes, with a single amino acid substitution influencing tropism.

Conclusions:

• In situ spatial transcriptomics is a powerful tool for resolving AAV biodistribution and BBB traversal at high resolution.
• Identified novel AAV variants with specific tropism for CNS cell types, including inhibitory neurons.
• Provides a blueprint for capsid engineering to achieve precise cell subtype targeting in the CNS for gene therapy applications.