Davis Brian M, Humeau Laurent, Slepushkin Vladimir, Binder Gwendolyn, Korshalla Lauren, Ni Yajin, Ogunjimi E Oluwakemi, Chang Lan-Fei, Lu Xiaobin, Dropulic Boro
VIRxSYS Corp, 200 Perry Pky, Ste 1A, Gaithersburg, MD 20877, USA.
Blood. 2004 Jul 15;104(2):364-73. doi: 10.1182/blood-2003-07-2363. Epub 2004 Apr 1.
High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34(+) cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors.
在造血祖细胞（HPC）中很难实现高基因转移效率，但这对HPC基因治疗的治疗成功至关重要。与常用于研究的经离心的载体相反，使用经柱纯化的载体进行临床应用时，高效基因转移尤其具有挑战性。我们研究了使用临床适用方案和经柱纯化的慢病毒载体数量来提高转导的新方法。认识到腺苷5'-三磷酸（ATP）结合盒（ABC）转运蛋白与HPC生物学的关联，我们研究了转运蛋白抑制剂对转导的影响。我们发现ABC转运蛋白抑制剂维拉帕米可将从动员的外周血、骨髓和脐带血中分离出的CD34(+)细胞的转导效率提高2至6倍。维拉帕米还将人类重症联合免疫缺陷（SCID）重建造血细胞（SRC）转导提高了3至4倍，在接受初次和二次移植的小鼠中产生了80%至90%的转导水平，且多谱系重建未发生改变。其他ABC转运蛋白底物抑制剂如奎尼丁、地尔硫卓和利托那韦也将转导提高了2至3倍，尽管不是底物的ABC转运蛋白抑制剂则没有。在成熟造血细胞、神经球、间充质干细胞或肝细胞中未观察到转导增强。在水泡性口炎病毒G（VSV-G）假型慢病毒载体中观察到HPC中转导增强，但在RD114假型载体中未观察到。因此，我们提出了一种通过VSV-G假型慢病毒载体有效递送至原始HPC的新方法。
