Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH.
Fred Hutchinson Cancer Research Center, Seattle, WA.
Exp Hematol. 2020 Jun;86:28-42.e3. doi: 10.1016/j.exphem.2020.05.009. Epub 2020 May 27.
As important vectors for ectopic protein expression, gene silencing, and progenitor cell barcoding, lentiviruses continue to emerge as versatile research and clinical tools. For studies employing cell types that are relatively resistant to transduction, high-titer lentivirus preparations with low cytotoxicity are required. During lentivirus production, carryover plasmid DNA endotoxins, transfection reagents, damaged packaging cells, and virus concentration procedures are potential sources of cytotoxicity. As an often unevaluated property of lentivirus preparations, cytotoxicity can unwittingly skew estimates of functional titers and complicate interpretations of transduced cell phenotypes. By employing hematopoietic UT7epo cells cultured in erythropoietin (EPO) below maximal dosing, we first define a sensitive flow cytometric bioassay for critically assessing the cytotoxicity (and titers) of lentivirus preparations. Bioassay of custom preparations of research-grade lentiviruses from six commercial sources unexpectedly revealed substantial cytotoxicity (with certain preparations additionally registering titers several log below designated values). To overcome such limiting properties, we further report on unique, efficient workflows for reproducibly preparing and processing high-titer, low-cytotoxicity (HTLC) lentiviruses at research scale. These HTLC lentiviruses reliably transduce peripheral blood hematopoietic stem/progenitor cells (PB-HSPCs) at frequencies ≥40%, with low cytotoxicity. In addition, by employing cyclosporin H (to inhibit IFITM3), PB-HSPCs can be transduced at heightened efficiency with nominal cytotoxicity. Overall, this work provides straightforward approaches to (1) critical assessment of the cytotoxicity of lentivirus preparations; (2) reproducible generation (and concentration) of high-quality lentiviruses via a streamlined workflow; and (3) transduction of PB-HSPCs at benchmark levels with nominal cytotoxicity.
作为异位蛋白表达、基因沉默和祖细胞标记的重要载体，慢病毒继续成为多功能的研究和临床工具。对于使用对转导相对具有抗性的细胞类型的研究，需要具有低细胞毒性的高滴度慢病毒制剂。在慢病毒生产过程中，残留质粒 DNA 内毒素、转染试剂、受损包装细胞和病毒浓缩程序都是潜在的细胞毒性来源。作为慢病毒制剂的一个经常未被评估的特性，细胞毒性可能会无意识地影响功能滴度的估计，并使转导细胞表型的解释复杂化。通过使用在最大剂量以下接受促红细胞生成素 (EPO) 培养的造血 UT7epo 细胞，我们首先定义了一种敏感的流式细胞术生物测定法，用于严格评估慢病毒制剂的细胞毒性（和滴度）。对来自六个商业来源的定制研究级慢病毒制剂的生物测定出人意料地显示出显著的细胞毒性（某些制剂的滴度还记录了几个对数低于指定值）。为了克服这种限制性质，我们进一步报告了独特、高效的工作流程，用于在研究规模上可重复地制备和处理高滴度、低细胞毒性 (HTLC) 慢病毒。这些 HTLC 慢病毒以 ≥40%的频率可靠地转导外周血造血干/祖细胞 (PB-HSPC)，且具有低细胞毒性。此外，通过使用环孢菌素 H（抑制 IFITM3），可以在名义细胞毒性下以提高的效率转导 PB-HSPC。总体而言，这项工作提供了简单的方法来：(1) 对慢病毒制剂的细胞毒性进行关键评估；(2) 通过简化的工作流程可重复地生成（和浓缩）高质量的慢病毒；(3) 在名义细胞毒性下以基准水平转导 PB-HSPC。
