Single Particle Adsorption and Response Quantification for Functional AAVx Titer and Dose Analysis

Abstract

Accurate quantification of functional adeno-associated virus (AAV) dose remains a critical limitation in gene therapy, where titers are defined using ensemble measurements of viral genomes or capsid concentration that average across heterogeneous populations and weakly predict transduction. We present single-particle adsorption and response quantification (SPARQ), a diffraction-limited imaging platform for quantifying AAVx vectors, regardless of serotype, at single-nanoparticle and single-cell resolution. SPARQ immobilizes and resolves individual AAVx, enabling concurrent measurement of capsid-associated and genome-associated signals before cell exposure. Across multiple serotypes, including AAV2, AAV5, and AAVrh74, SPARQ identifies full-capsid fractions, consistent with bulk measurements obtained by ddPCR/ELISA, DLS/UV-Vis, charge detection, and mass photometry, while reporting narrower distributions than ensemble-derived values and revealing systematic discrepancies in bulk-derived titers arising from population averaging. Surface immobilization is achieved by charge-dependent AAVx-surface interactions, as supported by multiphysics simulations, which capture serotype-dependent adsorption kinetics governed by capsid electrostatics for AAV2, AAV5, AAV6, and AAV9. Furthermore, SPARQ directly measures transduction as a function of particle number and reveals a threshold response. Transduction efficiency increased from [~]29% (S.D {+/-} 3.31%) at [~]18 (S.D {+/-} 7.55) AAVs per cell to [~]66% (S.D {+/-} 1.66%) at [~]42 (S.D {+/-} 9.69) AAVs per cell, which is comparable with conventional in vitro assay requiring 105 AAVs/cell. Cumulative fluorescence measurements across large micro-patterned arrays of single cells recapitulate bulk-like high-throughput scaling, while SPARQs single-nanoparticle resolution reveals functional heterogeneity that is masked by population averaged assays. SPARQ provides a particle-resolved framework for functional AAVx titration and quantitative characterization, enabling the development of quality control and dosing strategies in gene therapy manufacturing. TeaserSPARQ combines light-activated surface viral adsorption with diffraction-limited imaging to directly quantify AAV integrity, heterogeneity, and functional transduction at the single-particle level.