Size-Controlled and Shelf-Stable DNA Particles for Production of Lentiviral Vectors

Yizong Hu, Yining Zhu, Nolan D. Sutherland, David R. Wilson, Marion Pang, Ester Liu, Jacob R. Staub, Cynthia A. Berlinicke, Donald J. Zack, Jordan J. Green, Sashank K. Reddy, Hai Quan Mao

Research output: Contribution to journalArticlepeer-review

Abstract

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 shelf-stable 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.

Original languageEnglish (US)
Pages (from-to)5697-5705
Number of pages9
JournalNano Letters
Volume21
Issue number13
DOIs
StatePublished - Jul 14 2021

Keywords

  • kinetic growth
  • lentiviral vector production
  • particle size
  • plasmid DNA
  • poly(ethylenimine)
  • transfection

ASJC Scopus subject areas

  • Bioengineering
  • Chemistry(all)
  • Materials Science(all)
  • Condensed Matter Physics
  • Mechanical Engineering

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