Duvshani-Eshet Maayan, Keren Hadas, Oz Shira, Radzishevsky Inna S, Mor Amram, Machluf Marcelle
The Faculty of Biotechnology and Food Engineering, The Technion-Israel institute of Technology, Haifa Israel.
J Gene Med. 2008 Oct;10(10):1150-9. doi: 10.1002/jgm.1235.
One of the major limitations of nonviral gene delivery methods is nuclear transport of plasmid DNA (pDNA). Peptides bearing nuclear localization signal (NLS) were shown to mediate nuclear import of macromolecules. We have explored the use of cell-permeable peptides (CPP) bearing NLS sequences to enhance transfection mediated by a nonviral approach: therapeutic ultrasound (TUS).
Two CPP-NLS peptides which differ in the location of the NLS relative to the CPP were used: S4 13-PV and PV-S4 13. The peptides were attached to pDNA using electrostatic interactions. Gel-electrophoresis and fluorescent assays were performed to evaluate pDNA-peptide interactions and condensation effects. Confocal microscopy was used to evaluate pDNA-peptide interaction inside cells. Transfection studies were conducted with the luciferase gene, using pDNA-peptides alone, or with the application of TUS.
Attachment of both peptides to pDNA condensed the pDNA, with higher affinity for the S4(13)-PV peptide. This interaction protected pDNA from endonucleases, but was also reversible. Both peptides mediated pDNA delivery to cell cytoplasm, but less significantly to the nucleus. Thus, both peptides produced transfection in cells, when added after incubation with DNA, with higher transfection-level for PV-S4 13. Application of TUS increased transfection mediated by these peptides, but was not higher compared to transfection using TUS and pDNA alone.
This study suggests that CPP-NLS peptides may be used for condensing pDNA and bringing it into the cell cytoplasm, but their ability to mediate nuclear import of pDNA is insignificant.
非病毒基因递送方法的主要局限性之一是质粒DNA（pDNA）的核转运。带有核定位信号（NLS）的肽已被证明可介导大分子的核输入。我们探索了使用带有NLS序列的细胞穿透肽（CPP）来增强非病毒方法——治疗性超声（TUS）介导的转染。
使用了两种NLS相对于CPP位置不同的CPP-NLS肽：S4 13-PV和PV-S4 13。通过静电相互作用将肽连接到pDNA上。进行凝胶电泳和荧光测定以评估pDNA-肽相互作用和凝聚效果。共聚焦显微镜用于评估细胞内的pDNA-肽相互作用。使用荧光素酶基因进行转染研究，单独使用pDNA-肽，或应用TUS。
两种肽与pDNA的连接都使pDNA凝聚，对S4(13)-PV肽具有更高的亲和力。这种相互作用保护pDNA免受核酸内切酶的作用，但也是可逆的。两种肽都介导pDNA递送至细胞质，但进入细胞核的效率较低。因此，当与DNA孵育后添加时，两种肽都能在细胞中产生转染，PV-S4 13的转染水平更高。TUS的应用增加了这些肽介导的转染，但与单独使用TUS和pDNA转染相比并没有更高。
本研究表明，CPP-NLS肽可用于凝聚pDNA并将其带入细胞质，但其介导pDNA核输入的能力微不足道。
