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A Longitudinal 3D Live-Cell Imaging Platform to Uncover AAV Vector-Host Dynamics at Single-Cell Resolution
Marlies Leysen1, Nicolas Peredo2,3, Benjamin Pavie2,3,4
1Trellis Research Group, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
International Journal of Molecular Sciences
|January 10, 2026
Summary
Researchers developed a new imaging method to track recombinant adeno-associated viral vectors (rAAVs) in cells. This study reveals how rAAVs move within cells, identifying key factors that improve gene delivery for better gene therapies.
Area of Science:
- Molecular Biology
- Cell Biology
- Gene Therapy
Background:
- Recombinant adeno-associated viral vectors (rAAVs) are crucial for gene therapy but face challenges in efficient nuclear delivery.
- Understanding rAAV subcellular trafficking is essential for improving gene transduction efficiency.
Purpose of the Study:
- To develop and utilize a novel live-cell imaging and analysis platform to investigate rAAV2 trafficking dynamics.
- To identify factors influencing rAAV nuclear delivery and transgene expression.
Main Methods:
- Established a longitudinal confocal live-cell imaging workflow to track rAAV2 from 4 to 12 hours post-transduction.
- Developed an automated 3D analysis pipeline for quantifying spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution.
- Evaluated the impact of vector dose, cell cycle, and empty particles on rAAV trafficking and transduction.
Main Results:
- Identified novel trafficking features associated with high transgene expression.
- Demonstrated that higher rAAV2 doses and cell cycle progression enhance cytoplasmic trafficking, nuclear delivery, and transgene expression.
- Characterized empty rAAV2 particles, showing distinct trafficking patterns and significantly lower nuclear accumulation compared to genome-containing vectors.
Conclusions:
- The developed platform provides mechanistic insights into rAAV transduction bottlenecks.
- Findings offer potential strategies for optimizing AAV-based gene therapy by improving vector delivery.
- The platform's design is generalizable for studying other non-enveloped viruses.
