Spatial transcriptomics for profiling the tropism of viral vectors in tissues

Area of Science:

• Molecular Biology
• Neuroscience
• Biotechnology

Background:

• Advancing engineered adeno-associated viral vectors (AAVs) for precise cell subtype targeting requires high-throughput, high-resolution in vivo transduction profiling assays.
• Current methods lack the resolution and throughput to fully characterize AAV tropism in complex biological systems.

Purpose of the Study:

• To develop an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA.
• To characterize the in vivo transduction profiles and cell subtype tropisms of systemically administered AAV variants in the mouse brain.

Main Methods:

• Developed USeqFISH by integrating hydrogel-based tissue clearing, enhanced signal amplification, and sequential labeling for multiplexed RNA detection.
• Applied USeqFISH to investigate transduction patterns of six systemic AAVs, including the novel AAV-PHP.AX variant, across various mouse brain regions.
• Demonstrated USeqFISH's capability for profiling pooled regulatory cargos and its potential for non-human primate studies.

Main Results:

• Revealed distinct cell subtype biases for each AAV variant, identifying specific tropisms for neurons and astrocytes.
• Identified AAV-PHP.N exhibiting a bias toward excitatory neurons.
• Successfully profiled a 13-variant pool of microRNA target sites within AAV genomes using USeqFISH.

Conclusions:

• USeqFISH provides a powerful tool for high-resolution spatial transcriptomic profiling of AAV transduction in intact tissues.
• The method enables detailed characterization of AAV tropism, crucial for optimizing gene delivery strategies.
• USeqFISH holds promise for in situ AAV profiling and multimodal single-cell analysis in preclinical models and potentially in non-human primates.