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Concurrent measures of fusion and transduction efficiency of primary CD34+ cells with human immunodeficiency virus 1-based lentiviral vectors reveal different effects of transduction enhancers

机译:同时检测基于人免疫缺陷病毒1的慢病毒载体对原代CD34 +细胞融合和转导效率的方法揭示了转导增强子的不同作用

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摘要

Lentivirals vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of non-replicative recombinant LVs in various cell types, including primary human hematopoietic stem/progenitor cells (HSPCs), and to quantify the level of transduction of the same initially-infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers like Vectofusin-1 or Retronectin can partially relieve the postentry block but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols.
机译:慢病毒载体(LVs)用于各种基因转移应用,特别是用于造血基因治疗,但是缺乏准确评估与载体进入靶细胞有关的控制转导效率的参数的方法。我们采用了基于荧光共振能量转移的人类免疫缺陷病毒1融合测定法,以测量非复制型重组LV在各种细胞类型(包括原代人类造血干/祖细胞(HSPC))中的进入,并量化转导水平相同的最初感染的细胞。该测定法利用包含β-内酰胺酶(BLAM)-Vpr嵌合蛋白(BLAM-LV)并编码截短形式的低亲和力神经生长因子受体(ΔNGFR)的重组LV。用BLAM-LV感染靶细胞后,载体进入会迅速导致BLAM-Vpr释放到细胞质中,这可以通过使用流式细胞仪切割荧光底物来测量。相同感染细胞的平行培养显示出ΔNGFR表达导致的转导效率。这种基于LV的融合/转导测定是一种动态且多功能的工具,例如揭示了已知在造血起源的细胞,特别是人HSPC中发生的LV的进入后限制。此外,这种BLAM-LV分析使我们能够评估HSPCs的细胞因子预刺激对LVs进入步骤的影响。该测定还表明,转导增强剂(如Vectofusin-1或Retronectin)可以部分缓解进入后阻滞,但它们在促进LV进入方面的作用不同。总之,一种这样的测定法对于研究针对左心室的造血后进入限制应是有用的,因此应允许改进各种基于左心室的基因治疗方案。

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