title: Enhanced non-viral gene delivery by coordinated endosomal release and inhibition of β-tubulin deactylase.
abstract: Efficient non-viral gene delivery is highly desirable but often unattainable with some cell-types. We report here that non-viral DNA polyplexes can efficiently transfect differentiated neuronal and stem cells. Polyplex transfection centrifugation protocols was enhanced by including a simultaneous treatment with a DOPE/CHEMS lipid suspension and a microtubule inhibitor, Tubastatin A. Lipoplex transfection protocols were not improved by this treatment. This mechanism of action was unravelled by systematically identifying and rationally mitigating barriers limiting high transfection efficiency, allowing unexpected improvements in the transfection of mesenchymal stem cells (MSC), primary neuron and several hard-to-transfect cell types beyond what are currently achievable using cationic polymers. The optimized formulation and method achieved high transfection efficiency with no adverse effects on cell viability, cell proliferation or differentiation. High efficiency modification of MSC for cytokine overexpression, efficient generation of dopaminergic neuron using neural stem cells and enhanced genome editing with CRISPR-Cas9 were demonstrated. In summary, this study described a cost-effective method for efficient, rapid and scalable workflow for ex vivo gene delivery using a myriad of nucleic acids including plasmid DNA, mRNA, siRNA and shRNA.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology, and Research (A*STAR), 138668 Singapore.
authors.forename: Yoon Khei
authors.initials: YK
authors.lastname: Ho
authors.affiliation: MiRXES, 10 Biopolis Road, Chromos 03-01, 138670 Singapore.
authors.forename: Li Han
authors.initials: LH
authors.lastname: Zhou
authors.affiliation: Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Kam C
authors.initials: KC
authors.lastname: Tam
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology, and Research (A*STAR), 138668 Singapore.|Department of Biochemistry, National University of Singapore, 119260 Singapore.
authors.forename: Heng Phon
authors.initials: HP
authors.lastname: Too
mesh_terms: D000107:Acetylation; D000818:Animals; D001692:Biological Transport; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D011992:Endosomes / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D058953:Neural Stem Cells / Q000166:cytology; D011108:Polymers / Q000737:chemistry; D014162:Transfection / Q000379:methods*; D014404:Tubulin / Q000378:metabolism*
chemical_list: D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D007211:Indoles; D011108:Polymers; D014404:Tubulin; C553587:tubastatin A; D004247:DNA