HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers
HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers
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DOI：
10.1089/108729003764097322
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发表时间：
2003-02-01
期刊：
影响因子：
--
通讯作者：
Iversen, PL
中科院分区：
文献类型：
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作者：
Moulton, HM;Hase, MC;Iversen, PL
磷酸二酯morpholino oligomers （PMO）是一种不带电的反义分子，结合RNA的互补序列，通过阻止翻译或干扰pre-mRNA剪接来抑制基因表达。将PMO送入培养细胞的技术主要是机械方法。这些交付方法虽然有用，但也有局限性。我们研究了HIV Tat肽（pTat）和其他阳离子肽将PMO传递到培养细胞中的能力。ptat - pmo -荧光素偶联物处理的HeLa细胞100%可见荧光。pTat-PMO偶联物靶向c-myc mRNA下调c-myc报告基因表达，IC50为25 muM，达到近100%的抑制作用。pTat-PMO偶联物靶向β -珠蛋白前mrna剂量依赖性纠正剪接的突变剪接位点，并上调功能性报告基因的表达。未偶联的PMO和未偶联的pTat均未引起反义活性。然而，与机械介导的PMO传递相比，ptat介导的PMO传递需要更高浓度的PMO （bbb10mum）才能引起反义活性并引起一定的毒性。大多数pat - pmo偶联物与细胞膜相关，内化偶联物定位于囊泡、细胞质和细胞核。其他三种阳离子肽的效果远不如pTat。pTat在100%的被测细胞中显著提高PMO的递送。ptat介导的交付是一个比其他交付方法简单得多的过程。
Phosphorodiamidate morpholino oligomers (PMO) are uncharged antisense molecules that bind complementary sequences of RNA, inhibiting gene expression by preventing translation or by interfering with pre-mRNA splicing. The techniques used to deliver PMO into cultured cells have been mostly mechanical methods. These delivery methods, although useful, have limitations. We investigated the ability of the HIV Tat peptide (pTat) and other cationic peptides to deliver PMO into cultured cells. Fluorescence was seen in 100% of HeLa cells treated with pTat-PMO-fluorescein conjugate. pTat-PMO conjugate targeted to c-myc mRNA downregulated c-myc reporter gene expression with an IC50 of 25 muM and achieved nearly 100% inhibition. pTat-PMO conjugate targeted to a mutant splice site of beta-globin pre-mRNA dose-dependently corrected splicing and upregulated expression of the functional reporter gene. Neither unconjugated PMO nor unconjugated pTat caused antisense activities. However, compared with mechanically mediated delivery, pTat-mediated PMO delivery required higher concentrations of PMO (>10 muM) to cause antisense activity and caused some toxicity. Most pTat-PMO conjugate was associated with cell membranes, and internalized conjugate was localized in vesicles, cytosol, and nucleus. The other three cationic peptides are much less effective than pTat. pTat significantly enhances delivery of PMO in 100% of cells assayed. pTat-mediated delivery is a much simpler procedure to perform than other delivery methods.