The gene therapy industry lacks an analytical method for AAV empty-to-full capsid characterization that combines speed, affordability, precision, and automation, comparable to the robust standards of protein therapeutics. Current methods, while effective, are either time-consuming, costly, or require specialized equipment and expertise, creating a critical need for a purpose-built technology to streamline manufacturing workflows.
A microfluidic chip-based CRISPR diagnostic platform offers a rapid, cost-effective, and precise solution for AAV empty-to-full capsid characterization by leveraging the programmable detection capabilities of Cas12a and fluorescence-based readouts. This technology enables multiplexed detection of AAV genome sequences, including synthetic ITR targets, with high sensitivity (~4.2 pM), minimal material requirements, and compatibility with simple, smartphone-based readers. Combined with washing-free D4 protein quantification assays, the platform could provide a comprehensive, automated analytical tool tailored for gene therapy manufacturing.
This technology has the potential to streamline gene therapy process development, manufacturing validation, and continuous QC characterization.
Successful implementation of this project could pave the way for an additional powerful technique in the biomanufacturing PAT toolbox.
The CRISPR/D4 lab-on-a-chip technology reduces AAV quantification time from several hours to just 30 minutes, eliminating reliance on costly PCR and ELISA workflows. By streamlining process development and cutting equipment and consumable needs, manufacturers can achieve up to 50–70% savings in analytical testing costs and accelerate time-to-market for gene therapies—translating into millions in annual operational savings and improved production efficiency.
CRISPR/D4 Empty-to-Full AAV Quantification Tool: A lab-on-a-chip platform combining CRISPR-based genome detection and D4 immunoassay for capsid quantification, enabling rapid and precise measurement of empty-to-full AAV ratios.
Prototype Assay Chips: Developed and validated CRISPR and D4 assay chips that maintain functionality after long-term storage and meet performance criteria in production-related environments.
Analytical Method Advancement: Demonstrated a low-cost, high-throughput alternative to conventional ddPCR and ELISA methods, reducing assay time to 30 minutes and simplifying workflows.
Hetzler, Z., Marinakos, S. M., Lott, N., Mohammad, N., Lass-Napiorkowska, A., Kolbe, J., Turrentine, L., Fields, D., Overton, L., Marie, H., Hucknall, A., Rammo, O., George, H., & Wei, Q. (2024). Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a. Gene Therapy, 31(5-6), 304-313. https://doi.org/10.1038/s41434-024-00449-x
Hetzler, Z., Marinakos., S., Loft, N., & Mohammad, N., A Platform CRISPR-Cas12a Assay for Rapid Quantification of Adeno-Associated Virus Vector Genome Titers, 2023 AIChE Annual Conference, Orlando, FL, November 6, 2023.
Hetzler, Z., Rapid Adeno-associated virus vector genome quantification with amplification-free CRISPR-Cas12a diagnostics, ACS Fall 2023 National Meeting, San Francisco, CA, August 13, 2023.
Wei, Q., Molecular Chip Device for Rapid AAV Full and Empty Vector Quantitation, 2023 NIIMBL National Meeting, Washington, DC, June 29, 2023.
Wei, Q., Multifunctional Microfluidic Chip for Rapid Vector Genome and Empty Capsid Quantitation in AAV Vector Production (PC4.1-116), NIIMBL Member Forum, Virtual, June 30, 2022.
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North Carolina State University
Biostealth Inc.
EMD Millipore Corporation
