NANO-PAVE: A Nanopore-Based Method for AAV Capsid Quality Assessment

About This Grant

Adeno-associated viruses (AAVs) are the leading vectors for gene therapy, valued for their safety, low immunogenicity, and broad tissue tropism. However, large-scale clinical translation remains hindered by challenges in manufacturing quality control. A critical barrier is the inability to rapidly and reliably measure capsid load and the ratio of full to empty capsids—key attributes that directly affect therapeutic potency and safety. Current assays such as ELISA (capsid titer), ddPCR (genome titer), and AUC (full/empty ratio) are costly, labor-intensive, and serotype-dependent, leading to inconsistent results across laboratories and production runs. To overcome these limitations, we will develop NANO-PAVE (NANOpore-based analysis of Proportion of full and empty Adeno-associated Virus capsids via nanopore Evaluation), a solid-state nanopore platform that analyzes individual AAV particles without labels, dyes, or antibodies. By leveraging voltage-driven translocation and electro-deformation, NANO-PAVE directly links mechanical signatures to genome packaging state, enabling real-time, quantitative discrimination between full and empty capsids. In addition, its single-particle resolution will allow accurate measurement of capsid load across serotypes, providing a powerful complement to existing bulk assays. This approach is cost-effective, scalable, and adaptable to all serotypes, making it ideally suited for integration into manufacturing pipelines. Phase I will establish feasibility by demonstrating robust particle discrimination, validating nanopore- derived measurements against orthogonal benchmarks, exploring genome-structure effects on capsid mechanics, and testing multiple AAV serotypes to ensure broad applicability of NANO-PAVE across vector types. Phase I deliverables will include a validated serotype-independent NANO-PAVE technology—integrating optimized nanopore design, recapture protocol, and machine-learning analysis—benchmarked against established assays to provide a rigorous, quantitative QC approach. This work will lay the foundation for Phase II development of a scalable, in-process analytical tool capable of distinguishing therapeutically relevant capsids from product-related impurities (empty, partially filled capsids, aggregates) and process-related impurities such as host cell proteins and media components, enabling comprehensive quality assessment. Ultimately, NANO-PAVE has the potential to become a transformative QC platform for academic, clinical, and industrial gene therapy programs, improving safety, efficacy, and manufacturing efficiency.