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Related Experiment Video

Updated: Jan 13, 2026

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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
PubMed
Summary
This summary is machine-generated.

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.

Keywords:
automated 3D image analysislive-cell imagingrecombinant AAVviral trafficking

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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.