Cationic Liposome-Mediated CXCR4 Gene Delivery into Hematopoietic Stem/Progenitor Cells: Implications for Clinical Transplantation and Gene Therapy
Cationic Liposome-Mediated CXCR4 Gene Delivery into Hematopoietic Stem/Progenitor Cells: Implications for Clinical Transplantation and Gene Therapy
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DOI：
10.1089/scd.2011.0297
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发表时间：
2012-07-01
影响因子：
4
通讯作者：
Chen, Jie
中科院分区：
文献类型：
--
作者：
Gul-Uludag, Hilal;Xu, Peng;Chen, Jie
趋化因子基质细胞衍生因子(SDF)-1α/CXCL12及其受体CXC趋化因子受体4(CXCR4)在造血干/祖细胞(HSPC)在骨髓中的归巢/植入和保持中起重要作用。病毒基因转移技术已经证明，CXCR4在人CD34(+)HSPC上的过表达显著改善了它们在小鼠模型中的植入。然而，基因治疗的临床试验显示，由于病毒基因随机整合到宿主基因组中，与病毒携带者的免疫原性有关的安全问题。因此，需要一种安全、无病毒、高效的方法将CXCR4基因导入HSPC，以提高临床移植和基因治疗水平。在本工作中，我们用阳离子脂质体IBAfect研究了非病毒CXCR4基因进入HSPC的情况。我们使用了脐血CD34(+)细胞和表达CD34抗原的未成熟造血细胞模型，即白血病细胞系KG-1a和KG-1。分别于转染后12、24、48、72 h用流式细胞仪检测细胞转染率，台盼蓝拒染法和MTS法检测细胞存活率。趋化实验检测CXCR4基因转染的HSPC对SDF-1α梯度的功能反应。我们发现，KG-1a和KG-1细胞的转染率接近25%，HSPC的转染率为20%，而CXCR4基因的转染率对HSPC的存活率没有显著影响。更重要的是，使用IBAfect过表达CXCR4显著增加了KG-1细胞和HSPC对SDF-1α的趋化能力。然而，我们平行测试了另外两个商用阳离子脂质体(Lipoect tamine2000和1，2-二油酰基-3-三甲基丙烷[DOTAP])，发现它们在相同条件下未能将CXCR4基因转移到细胞中。这些结果表明，IBAfect介导的体外基因传递在HSPC上过表达CXCR4是一种安全有效的技术，在提高HSPC移植和基因治疗方案的有效性方面具有很大的潜力。
The chemokine stromal cell-derived factor (SDF)-1 alpha/CXCL12 and its receptor CXC chemokine receptor 4 (CXCR4) play a crucial role in the homing/engraftment and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. It has been shown using the viral gene transfer technique that CXCR4 overexpression on human CD34(+) HSPC significantly improves their engraftment in murine models. However, clinical trials with gene therapy have revealed safety concerns related to the immunogenicity of the viral carriers, due to the random integration of viral genes into the host genome. Therefore, a method for CXCR4 gene delivery into HSPC that is safe, nonviral, and highly efficient is needed to improve clinical transplantation and gene therapies. In this work, we investigated the nonviral CXCR4 gene delivery into HSPC using the cationic liposome agent IBAfect. We used CD34(+) cells from cord blood and the models of immature hematopoietic cells expressing CD34 antigen, namely, leukemic cell lines KG-1a and KG-1. Transfection efficiency was determined by flow cytometric analysis 12, 24, 48, and 72 h after transfection, and the viability of cells analyzed by trypan blue exclusion and MTS assays. The functional response of CXCR4-transfected HSPC toward an SDF-1 alpha gradient was determined by chemotaxis assay. We found that similar to 25% transfection is achieved for KG-1a and KG-1 cells and 20% for HSPC, and that the viability of CXCR4-transfected HSPC is not significantly altered. More importantly, overexpression of CXCR4 using IBAfect significantly increased the chemotaxis of KG-1 cells and HSPC toward SDF-1 alpha. However, we tested 2 other commercially available cationic liposomes (Lipofectamine 2000 and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) in parallel, and we found that they failed to deliver the CXCR4 gene into cells under the same conditions. These results suggest that IBAfect-mediated in vitro gene delivery to overexpress CXCR4 on HSPC is a safe and efficient technique with great potential for improving the efficacy of HSPC transplantation and gene therapy protocols.