Bioconjug Chem. 2019 Feb 20;30(2):418-431. doi: 10.1021/acs.bioconjchem.8b00760. Epub 2018 Dec 10.
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.
需要快速、高效且廉价的方法将功能性核酸递送至原代人细胞类型，以推进再生医学和细胞疗法。与常见的报告质粒（约 5-8 kbp）相比，基于质粒的基因编辑（如 CRISPR-Cas9）可能需要递送较大的质粒（约 9.5-13 kbp）。为了开发更有效的质粒递送载体，我们使用经肝素处理的含海藻糖的聚阳离子（Tr4-肝素）研究了质粒大小对原代人真皮成纤维细胞（HDFs）和诱导多能干细胞（iPSCs）转染的影响。用 4.7 kbp 至 10 kbp 质粒进行转染时，两种质粒大小均显示出高比率的多聚物内化。然而，在 HDFs 中转染大质粒几乎被消除，在 iPSCs 中转染显著减少。使用分子添加剂来探测转染的细胞内障碍。用氯喹处理来破坏内涵体，用地塞米松和胸苷来破坏核膜。破坏核膜导致大质粒转染显著增加，表明大质粒的核定位可能更困难。为了证明这种制剂的潜在临床应用，用地塞米松-Tr4-肝素多聚物处理 HDFs 和 iPSCs，多聚物编码 dCas9-VP64、合成转录激活物，靶向 VII 型胶原。与未转染的对照相比，这些转染使 HDFs 和 iPSCs 中的胶原蛋白表达分别增强了 5 倍和 20 倍，比 Lipofectamine 2000 对照更有效。通过核破坏可以显著提高功能性质粒的转染效率，这可能会导致非病毒载体的开发得到改进，用于体外 CRISPR-Cas9 基因编辑。
