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Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells
journal contribution
posted on 2018-12-10, 19:18 authored by William
S. Boyle, Kirk Twaroski, Emily C. Woska, Jakub Tolar, Theresa M. ReinekeFast, efficient, and inexpensive
methods for delivering functional
nucleic acids to primary human cell types are needed to advance regenerative
medicine and cell therapies. Plasmid-based gene editing (such as with
CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5–13
kbp) in comparison to common reporter plasmids (∼5–8
kbp). To develop more efficient plasmid delivery vehicles, we investigated
the effect of plasmid size on the transfection of primary human dermal
fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using
a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections
with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization
with both plasmid sizes. However, transfection with the large plasmid
was nearly eliminated in HDFs and significantly reduced in iPSCs.
Molecular additives were used to probe intracellular barriers to transfection.
Chloroquine treatments were used to destabilize endosomes, and dexamethasone
and thymidine were used to destabilize the nuclear envelope. Destabilizing
the nuclear envelope resulted in significantly increased large-plasmid-transfection,
indicating that nuclear localization may be more difficult for large
plasmids. To demonstrate the potential clinical utility of this formulation,
HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes
encoding dCas9-VP64, synthetic transcription activator, targeted to
collagen type VII. These transfections enhanced collagen expression
in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an
untransfected control and were the more effective than the Lipofectamine
2000 control. Functional plasmid transfection efficiency can be significantly
improved by nuclear destabilization, which could lead to improved
development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.
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Functional plasmid transfection efficiencynonviral vehiclesheparin-treated trehalose-containing polycationcell therapiesadvance regenerative medicine4.7 kbpEnhance Glycopolymer-Mediated TransfectionPrimary Human FibroblastsHDFPlasmid-based gene editingprobe intracellular barriersplasmid delivery vehiclestranscription activatorplasmid sizespolyplex internalizationuntransfected control10 kbp plasmidsMolecular AdditivesInduced Pluripotent Stem Cells FastLarge PlasmidsMolecular additivesiPSCplasmid sizeChloroquine treatmentsLipofectamine 2000 controlFunctional CRISPR-Cas 9 Transcription Activation Ex Vivodexamethasone-Tr 4-heparin polyplexes encoding dCas 9-VPcell typescollagen type VIITr 4-heparin Transfectionscollagen expressionvivo CRISPR-Cas 9 gene editing
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