부산대학교 도서관

Kathryn M Luly,1,2 Huilin Yang,2,3 Stephen J Lee,2,3 Wentao Wang,1,2 Seth D Ludwig,2,3 Haley E Tarbox,4 David R Wilson,1,2,5 Jordan J Green,1– 3,5– 10 Jamie B Spangler1– 3,6,8 1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA; 4Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA; 5Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD, USA; 6Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 7Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 8Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 9Departments of Neurosurgery and Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 10Department of Materials Science & Engineering, Johns Hopkins University, Baltimore, MD, USACorrespondence: Jamie B Spangler, Departments of Biomedical Engineering and Chemical & Biomolecular Engineering, Johns Hopkins University, 400 N Broadway, Smith 5011, Baltimore, MD, 21231, USA, Tel +1 443 287-1708, Email jamie.spangler@jhu.edu Jordan J Green, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 400 N Broadway, Smith 5017, Baltimore, MD, 21231, USA, Tel +1 410 614-9113, Email green@jhu.eduPurpose: Transient transfection is an essential tool for recombinant protein production, as it allows rapid screening for expression without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and the resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of a class of cationic polymers, poly(beta-amino ester)s (PBAEs), as reagents for transient transfection in comparison to linear 25 kDa PEI, a commonly used transfection reagent.Methods: Transfection efficiency and protein production were assessed in human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions using PBAE-based nanoparticles in comparison to linear 25 kDa PEI. Production of both a cytosolic reporter and secreted antibodies was investigated.Results: In both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and enhanced production of a cytosolic reporter compared to linear 25 kDa PEI. This result extended to secreted proteins, as a model PBAE increased the production of 3 different secreted antibodies compared to linear 25 kDa PEI at culture scales ranging from 20 to 2000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticle formulation led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types.Conclusion: These results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.Keywords: non-viral gene transfer, transient transfection, mammalian cell expression systems, DNA delivery