Performance Period: 9/1/2021 to 8/31/2023
Gene therapy holds transformative potential for the treatment of numerous genetic disorders. Adeno associated viral (AAV) vectors are the most promising gene therapy delivery method and have seen a meteoric rise over the past decade in the biopharmaceutical industry. AAV production, however, lacks innovative process analytical technology (PAT) needed for continued success. A prominent analytical challenge is the characterization of the ratio of empty-to-full AAV capsids, the determination of which is required by the FDA. To this end, we propose a dualfunctional microfluidic chip utilizing CRISPR-based DNA quantification and a washing-free sandwich assay for at-line capsid quantification in a miniaturized format, delivering fast, reliable, and accurate determination of the empty-to-full capsid ratio and total vector genome (vg) on a single device. The CRISPR technology will quantify vg titer, and the sandwich assay will measure capsid titer. Together, the two miniature assays will yield an empty-to-full ratio with a much faster turnaround time compared to the existing methods. This technology has the potential to be adopted as a novel go-to characterization method for simultaneous detection of nucleic acid and protein targets on the same device and could be a vanguard for future on-line AAV PAT and CRISPR diagnostics.
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.
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North Carolina State University