Biology subjects

Kwak, K. J.

Publications and source records attributed to Kwak, K. J..

3 recordsLinked to original sources

bioRxiv2026

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

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.

bioengineering↗

bioRxiv2022

Engineering a Single Extracellular Vesicle Protein and RNA Assay (siEVPRA) via In Situ Fluorescence Microscopy in a UV Micropatterned Array

The physical and molecular heterogeneity of extracellular vesicles (EVs) confounds bulk biomarker characterization, thus encouraging the development of novel assays capable of profiling EVs at a single-vesicle resolution. Here, we present a single EV (siEV) protein and RNA assay (siEVPRA) to simultaneously detect proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs) in siEVs. The siEVPRA consists of an array of microdomains embedded on a polyethylene glycol (PEG)-coated glass surface produced via UV photopatterning, functionalized with antibodies to target siEV subpopulations. Fluorescently labeled antibodies and RNA-targeting molecular beacons (MBs) were used to generate signals for proteins, mRNAs, and miRNAs on siEVs detected by total internal reflection fluorescence microscopy (TIRFM), outperforming the sensitivities of ELISA and PCR by three orders of magnitude. Using the siEVPRA, we analyzed EVs harvested from glioblastoma (GBM) cell lines and demonstrated vesicular heterogeneity in protein, mRNA, and miRNA expression through colocalization analyses, and validated the results by bulk RNA sequencing. We further demonstrated the clinical utility of the siEVPRA by detecting different mRNAs and miRNAs associated with GBM in patient samples. Together, these results indicate that the siEVPRA provides an effective platform to investigate the heterogeneity of proteins and RNAs in subpopulations of EVs.

bioengineering↗

bioRxiv2021

An Immunogold Single Extracellular Vesicular RNA and Protein (AuSERP) Biochip to Predict Responses to Immunotherapy in Non-Small Cell Lung Cancer Patients

Conventional PD-L1 immunohistochemical tissue biopsies only predict 20~40% of non-small cell lung cancer (NSCLC) patients that will respond positively to anti-PD-1/PD-L1 immunotherapy. Herein, we present an immunogold biochip to quantify single extracellular vesicular RNA and protein (AuSERP) as a non-invasive alternative. With only 3 L of serum, PD-1/PD-L1 proteins on the surface of extracellular vesicles (EVs) and EV PD-1/PD-L1 messenger RNA (mRNA) cargo were detected at a single-vesicle resolution and exceeded the sensitivities of ELISA and qRT-PCR by 1000 times. By testing a cohort of 27 non-responding and 27 responding NSCLC patients, AuSERP indicated that the single-EV mRNA biomarkers surpass the single-EV protein biomarkers at predicting patient responses to immunotherapy. Dual single-EV PD-1/PD-L1 mRNA detection differentiated responders from non-responders with an accuracy of 72.2% and achieved an NSCLC diagnosis accuracy of 93.2%, suggesting the potential for AuSERP to provide enhanced immunotherapy predictions and cancer diagnoses within the clinical setting.

immunology↗