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

Abstract

Fast, 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.

Polymeric Delivery of Therapeutic Nucleic Acids

“…299 Dexamethasone dilates the nuclear pores, which can also promote the nuclear uptake of large plasmid DNA and increase the transfection efficiency. 302,328,329 For researchers endeavoring to improve the nuclear uptake of DNA cargo with polymer-based vehicles, it would be helpful to understand what levels of nuclear uptake are typically achieved with standard PEI-based transfections. Using quantitative polymerase chain reaction measurements, Szoka et al detected as few as 75 and as many as 50 000 plasmid copies (<5% of the applied dose) in the nuclei of transfected cells but found that levels above 3000 plasmids/nuclei yielded marginal returns in transgene expression.…”

Novos sistemas de delivery, uso tecnológico e reposicionamento da cloroquina e hidroxicloroquina: Uma revisão integrativa

“…No entanto, esta estratégia não tem se mostrado muito bem-sucedida em estudos clínicos, sendo um dos motivos alegados a falta de vetores seguros e eficientes para entregar genes exógenos terapêuticos às células alvos dos pacientes (Kay et al, 2001). A utilização de novos métodos para entrega celular de material genético exógeno possibilita o tratamento de diversas doenças adquiridas ou hereditárias, através do reparo ou substituição do gene defeituoso, resultando na modulação da expressão da proteína associada com a doença (Boyle et al, 2019;Tang et al, 2014).…”

“…Alguns trabalhos corroboram esses achados, uma vez que a CQ contribuiu para aumentar níveis de atividade de transfecção do polímero polietilenimina de baixo peso molecular (PLGE) (Deng et al, 2011). A CQ também foi utilizada para desestabilizar os endossomos, resultando em um aumento significativo da transfecção de plasmídeos grandes, o que pode levar a um melhor desenvolvimento de veículos não virais para edição de gene CRISPR-Cas9 ex vivo (Boyle et al, 2019).…”

Enhancers in polymeric nonviral gene delivery systems

“…Corticosteroid Dexamethasone can dilate the nuclear pores 70 . Reineke's group investigated the effect of dexamethasone on nuclear permeability of induced pluripotent stem cells (iPSCs) and primary human dermal fibroblasts (HDFs) 71 . They utilized dexamethasone to pretreat the cells in cultures before transfection and got a 16‐fold increase in transfection efficiency compared to carrier alone for the large pDNA transfection.…”