Journal
NANO LETTERS
Volume 21, Issue 13, Pages 5697-5705Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.1c01421
Keywords
plasmid DNA; poly(ethylenimine); transfection; lentiviral vector production; particle size; kinetic growth
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Funding
- bluebird bio, Inc.
- National Institutes of Health [NIBIB EB018358]
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A kinetics-based approach has been developed for assembling size-controlled polyelectrolyte complex particles from plasmid DNA and poly(ethylenimine), improving the production quality and consistency of lentiviral vectors for gene therapy. This new method not only streamlines the viral manufacturing process but also enhances production scalability and particle stability.
Polyelectrolyte complex particles assembled from plasmid DNA (pDNA) and poly(ethylenimine) (PEI) have been widely used to produce lentiviral vectors (LVVs) for gene therapy. The current batch-mode preparation for pDNA/PEI particles presents limited reproducibility in large-scale LVV manufacturing processes, leading to challenges in tightly controlling particle stability, transfection outcomes, and LVV production yield. Here we identified the size of pDNA/PEI particles as a key determinant for a high transfection efficiency with an optimal size of 400-500 nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based approach to assemble size-controlled and shelfstable particles using preassembled nanoparticles as building blocks and demonstrated production scalability on a scale of at least 100 mL. The preservation of colloidal stability and transfection efficiency was benchmarked against particles generated using an industry standard protocol. This particle manufacturing method effectively streamlines the viral manufacturing process and improves the production quality and consistency.
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