Aug 2026· Journal of Visualized Experiments· Vol 234· 0 citations
Medicine
TL;DR
A streamlined three-dimensional spheroid platform optimized for evaluating AAV potency, transgene expression kinetics, and serotype-specific transduction efficacy across diverse cell lines is described and establishes its utility as a scalable and physiologically relevant system for preclinical gene therapy evaluation and development.
Abstract
Adeno-associated viruses (AAVs) are potent vectors used for gene delivery in gene therapy products. Their development requires in vitro systems that can reliably detect differences in vector design, serotype performance, regulatory element strength, and expression kinetics. These systems must also support applications such as potency assessment and vector optimization. Here, we describe a streamlined three-dimensional spheroid platform optimized for evaluating AAV potency, transgene expression kinetics, and serotype-specific transduction efficacy across diverse cell lines. Uniform spheroids are generated using ultra-low attachment plates and maintained under conditions that support stable architecture and long-term imaging. Following AAV transduction, fluorescent or luminescent readouts are monitored in real time using live-cell imaging systems. This enables quantitative assessment of reporter signal, dose responsiveness, regulatory element activity, and onset time through continuous kinetic imaging. The platform effectively discriminates between potent and weak vector genome designs and among multiple AAV serotypes. This method demonstrates robust performance across both slowly and rapidly dividing cell lines. These results establish its utility as a scalable and physiologically relevant system for preclinical gene therapy evaluation and development.
A novel approach to detarget liver transduction is developed by transiently downregulating the expression of key entry factors in this tissue using GalNac-siRNAs prior to AAV9 administration, which blunted hepatic transduction but also redirected the vector to other transduction-permissive tissues.
Katie Kubek-Luck, J. Velazquez, Xiao-Rui Yao et al.· Molecular Therapy· 0 citations
Evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing are synthesized.
Mariam Abdelnaby, A. Galiakberova, E. Dashinimaev· International Journal of Mol...· 0 citations
Adeno-associated virus (AAV) vectors are widely used for gene delivery, but inefficient transduction can require high vector doses. We tested whether unmodified linear polyvinylpyrrolidone (PVP), a pharmaceutical excipient, can improve AAV formulation without chemical modification of the vector or polymer. PVP10, PVP40, and PVP360 were evaluated in vitro across HEK293, HeLa, MEF, and CHO cells; selected formulations were tested after intravenous delivery, and 3% PVP40 was tested by subretinal delivery. In vitro, 1-3% PVP increased AAV8- and AAV9-mediated GFP expression across multiple cell lines. PVP360 showed broad activity in the initial cross-cell assay, whereas the HEK293 molecular-weight screen identified PVP40 and PVP360 as the most active formulations. The substantial fold increase observed in CHO cells largely reflected the low baseline transduction of the control group. MTT absorbance declined with concentrated PVP360, whereas PVP40 retained transduction-enhancing activity and was selected for local testing. In the HeLa AAV-DJ assay, the response pattern differed between MOI 1,000 and MOI 100; the largest observed increases in GFP-positive area occurred with 1.5% PVP10 and 1.5% PVP360 at MOI 100. Intravenous AAV9 delivery with PVP did not consistently increase ex vivo organ reporter signal. By contrast, subretinal delivery of AAV-PHP.eB with 3% PVP40 produced a 1.79- fold larger mean DsRed-positive area per retinal section, averaged within each eye (Welch’s t-test p = 0.063; Bayesian Pr[Δ > 0] = 0.952). These findings support further evaluation of PVP40 for local subretinal AAV delivery.
N. Gogoleva, T. Tran, Mayuko Oki et al.· bioRxiv· 0 citations
The data support the genomic safety of rAAV6 and its applicability to hematological gene therapy and functional enrichment analysis indicated associations with general cellular and structural processes, without enrichment in oncogenic pathways.
H. Lee, Nayoung Park, In-Byung Park et al.· International Journal of Ste...· 0 citations
This platform provides a powerful approach for engineering next-generation AAV vectors with customizable targeting profiles for diverse therapeutic applications and preserves AAV integrity and infectivity, and enables programmable retargeting of AAV tropism toward disease-relevant receptors.
Quan Pham, Jake Glicksman, Abhishek Chatterjee· Methods in molecular biology· 0 citations
Dual adeno-associated virus (AAV) vector reconstitution has become an indispensable technique for delivering large genes in neuroscience and gene therapy. A novel technology termed StitchR addresses the challenge of low efficiency in dual-AAV reconstruction, yet related tools enabling targeted gene delivery to specific neural cell populations remain insufficiently developed. Herein, we developed four StitchR-based dual-viral reconstitution strategies by combining high-efficiency AAV serotypes with neuron- or glia-specific promoters. Specifically, the AAV11-hSyn-driven StitchR strategy achieved specific transduction of local neurons and their upstream projection neurons (≥ 94.44% specificity); the AAV11-GfaABC1D-driven strategy realized astrocyte-specific transduction (approximately 98.25% specificity); and the AAV11-mIBA1-driven strategy enabled microglia-specific transduction (100% specificity). Furthermore, by incorporating the blood–brain barrier (BBB)-penetrating AAVhu.32-PLUS into the hSyn-driven system, we established a modified strategy that allows brain-wide neuronal transduction. Finally, we confirmed that the StitchR-based dual-viral reconstitution strategy can achieve efficient functional reconstitution of large transgenes in neural cells. Collectively, these dual-AAV delivery strategies expand the valuable AAV toolbox and offer crucial technical support for targeted delivery of large transgenes to defined neural cell populations, holding great significance for basic neuroscience research and potential therapeutic applications.
Nengsong Luo, Yunling Gao, Zengpeng Han et al.· Molecular Brain· 0 citations
