Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential
Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential
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DOI：
10.1038/sj.gt.3301859
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发表时间：
2003-01-01
期刊：
影响因子：
5.1
通讯作者：
Lee, KD
中科院分区：
文献类型：
--
作者：
Saito, G;Amidon, GL;Lee, KD
李斯特菌溶血素O(LLO)是单核细胞增生性李斯特氏菌的一种巯基激活的致孔蛋白，用于促进质粒DNA(PDNA)进入培养细胞的胞浆。为了赋予LLO对PDNA的络合能力，LLO的独特的半胱氨酸484通过可逆的、内体不稳定的二硫键与聚阳离子多肽鱼精蛋白(PN)以1：1的摩尔比偶联。巯基氧化的LLO结构LLO-S-S-Pn完全失去成孔活性，但还原后恢复了原有的活性。因此，使用该结构的增强胞液递送依赖于内源性细胞还原能力对LLO-S-S-PN中二硫键的必要还原。在HEK293、RAW264.7、P388D1细胞和有血清存在的骨髓巨噬细胞中，检测了含有LLO-S-S-PN的缩合PN/pDNA复合体对报告基因表达的增强作用。观察到具有不同重量比的所有测试络合物的显著增强。用LLO-S-S-PN代替1-4%的Pn时，在Pn/Pdna为0.64-0.80w/w时效果最为显著，导致荧光素酶的表达比Pn/PDNA高约三个数量级，且无明显毒性。这些结果表明，在基因递送策略中，以受控的方式将内溶LLO掺入PDNA递送系统是一种很有前途的提高靶细胞胞浆递送能力的方法。
Listeriolysin O (LLO), a sulfhydryl-activated pore-forming protein from Listeria monocytogenes, was tested and utilized for promoting plasmid DNA (pDNA) delivery into the cytosol of cells in culture. To render pDNA-complexing capability to LLO, the unique cysteine 484 of LLO was conjugated to polycationic peptide protamine (PN) at a 1: 1 molar ratio through a reversible, endosome-labile disulfide bond. The sulfhydryl-oxidized LLO construct, LLO-s-s-PN, completely lacked its pore-forming activity, yet regained its original activity upon reduction. The enhanced cytosolic delivery using this construct therefore relies on the requisite reduction of the disulfide, bond in LLO-s-s-PN by endogenous cellular reducing capacity. Condensed PN/pDNA complexes incorporating LLO-s-s-PN were tested for their enhanced gene delivery capability monitoring reporter gene expression in HEK293, RAW264.7, P388D1 cell lines and bone-marrow-derived macrophages in the presence of serum. Dramatic enhancement was observed for all tested complexes with varying weight ratios. The effect was most prominent at 0.64-0.80 (w/w) of PN/pDNA upon replacing 1-4% of PN with LLO-s-s-PN, resulting in approximately three orders of magnitude higher luciferase expression compared to PN/pDNA without apparent toxicity. These results demonstrate that incorporation of endosomolytic LLO into pDNA delivery systems in a controlled fashion is a promising approach of enhancing delivery into the cytosol of target cells in gene delivery strategies.