A Longitudinal 3D Live-Cell Imaging Platform to Uncover AAV Vector-Host Dynamics at Single-Cell Resolution

Sep 30, 2026

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Recombinant adeno-associated viral vectors (rAAVs) are leading gene delivery vehicles, but efficient nuclear delivery remains a major barrier due to incomplete knowledge of their subcellular trafficking dynamics. This work introduces a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, coupled with automated 3D image analysis quantifying vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. The platform evaluates vector dose, cell cycle progression, and empty particle behavior, revealing dose- and cell-cycle-dependent trafficking and previously undescribed pathways linked to high transgene expression, plus distinct empty-particle trafficking with reduced nuclear delivery. By identifying new bottlenecks in rAAV transduction, it provides mechanistic insights and generalizable strategies to improve AAV-based gene therapy.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages23

Reading Time58 min

AccessFree to download

Introduction

rAAV biology and barriers to transduction

Intracellular trafficking pathways

Dose dependence and mechanistic triggers

Article A Longitudinal 3D Live-Cell Imaging Platform to Uncover AAV Vector–Host Dynamics at Single-Cell Resolution Marlies Leysen 1, Nicolas Peredo 2,3, Benjamin Pavie 2,3,4, Benjamien Moeyaert 1 and Els Henckaerts 1,5, * Academic Editor: ZahraKadri Received: 28 November 2025 Revised: 17 December 2025 Accepted: 19 December 2025 Published: 25 December 2025 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. 1 Trellis Research Group, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium; [marlies.leysen@kuleuven.be](marlies.leysen@kuleuven.be) (M.L.); benjamien.moeyaert@kuleuven.be (B.M.) 2 VIB BioImaging Core Leuven, VIB Technologies, Center for Brain and Disease Research, B-3000 Leuven, Belgium; [nicolas.peredo@kuleuven.be](nicolas.peredo@kuleuven.be) (N.P.); benjamin.pavie@kuleuven.be (B.P.) 3 VIB BioImaging Core Leuven, Department of Neurosciences, KU Leuven, B-3000 Leuven, Belgium 4 VIB BioImaging Core Ghent, VIB Center for Inflammation Research, B-9052 Ghent, Belgium 5 Virus–Host Interactions & Therapeutic Approaches Research Group, Department of Microbiology, Immunology and Transplantation, KU Leuven, B-3000 Leuven, Belgium * Correspondence: [els.henckaerts@kuleuven.be](els.henckaerts@kuleuven.be) Abstract Recombinant adeno-associated viral vectors (rAAVs) are the leading gene delivery vehicles in clinical development, yet efficient nuclear delivery remains a major barrier to effective transduction. This limitation is partly due to the incomplete understanding of rAAV’s complex subcellular trafficking dynamics. Here, we establish a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, paired with an automated 3D analysis pipeline that quantifies spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. We use this platform to evaluate the effects of vector dose, cell cycle progression, and the behavior of empty particles. We identify previously undescribed trafficking features associated with high transgene expression. Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression facilitated both trafficking efficiency and transgene expression. We also characterize empty rAAV2 particles, revealing distinct trafficking patterns and markedly reduced nuclear accumulation compared to genome-containing vectors. By uncovering new bottlenecks in rAAV transduction, this platform provides mechanistic insights and potential strategies to improve AAV-based gene therapy. Its generalizable design further supports broad applicability to other nonenveloped viruses. Keywords: live-cell imaging; automated 3D image analysis; viral trafficking; recombinant AAV 1. Introduction Adeno-associated virus is a small (~25 nm), non-pathogenic parvovirus with a nonenveloped icosahedral capsid [1,2] . Its capsid is composed of three viral proteins—VP1, VP2, and VP3—in an approximate 1:1:10 ratio and encloses a linear single-stranded DNA genome. This genome contains two major open reading frames: rep, which encodes proteins required for viral replication, and cap, which encodes the structural capsid proteins, along with additional regulatory elements [3–5] . Recombinant AAV (rAAV) vectors are generated by replacing the native rep and cap coding sequences with a transgene of interest, enabling delivery of genetic material to host cells through the process of transduction. Over the past six decades, rAAV has emerged as the prime vector for in vivo gene therapy, primarily due to its non-pathogenic nature and capacity for stable, long-term expression across various tissues [6] . The clinical relevance of rAAV-based therapies is evident, with eight already approved treatments and

FAQs

What problem does the study address in rAAV gene delivery?

Efficient nuclear delivery is limited, largely because the subcellular trafficking dynamics of rAAV are not fully understood, reducing transduction efficiency.

How does the imaging platform in this study measure rAAV trafficking?

It uses a longitudinal confocal live-cell imaging workflow tracking rAAV2 from 4 to 12 hours post-transduction, combined with an automated 3D analysis pipeline to quantify vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution.

What did the study find about vector dose, cell cycle progression, and empty particles?

Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression supported both trafficking efficiency and transgene expression. Empty rAAV2 particles showed distinct trafficking patterns and markedly reduced nuclear accumulation compared with genome-containing vectors.

Sep 30, 2026

Upload by Jacob