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Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A.
Molecular Therapy - Methods & Clinical Development ( IF 4.6 ) Pub Date : 2019-11-15 , DOI: 10.1016/j.omtm.2019.11.003
Muhammad Elnaggar 1 , Anjud Al-Mohannadi 1 , Dhanya Kizhakayil 1 , Christophe Michel Raynaud 1 , Sharefa Al-Mannai 1 , Giusy Gentilcore 1 , Igor Pavlovski 1 , Abbirami Sathappan 1 , Nicholas Van Panhuys 1 , Chiara Borsotti 2 , Antonia Follenzi 2 , Jean-Charles Grivel 1 , Sara Deola 1
Affiliation  

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

中文翻译:

流式细胞术平台血细胞内FVIII的细胞内检测:一种新工具,以评估A型血友病的基因治疗效率。

通过流式细胞仪检测细胞中的因子VIII(FVIII)是有争议的,并且没有单克隆荧光抗体可商购。在这项研究中,我们优化了这种测定方法,并成功地将其用作研究磷酸甘油酸激酶(PGK)-FVIII慢病毒载体转导细胞的功能特性的平台,方法是在不同基因转移条件下直接观察细胞中的FVIII。通过关联基因表达和蛋白质积累数据,我们可以测量转导后的细胞应激参数。在转导的细胞系上进行的流式细胞仪分析显示,MOI速率提高会导致蛋白质水平升高,在MOI为30后趋于平稳。我们推测,在更高的MOI下,FVIII的产生可能会受到适当折叠所需的限制因素的影响。为了检验这个假设,我们通过阻止蛋白酶体降解来干扰未折叠的蛋白质反应,并测量细胞内错误折叠的蛋白质的积累。有趣的是,在更高的MOI值下,细胞显示出具有反应性氧种积累的毒性迹象。这表明需要确定安全的转导剂量窗口,以避免随后的细胞毒性。在这里,我们表明我们的流式细胞仪平台用于胞浆内FVIII蛋白检测是一种可靠的方法,可通过揭示基因转移后细胞的功能状态来优化血友病A中的基因治疗方案。这表明需要确定安全的转导剂量窗口,以避免随后的细胞毒性。在这里,我们表明我们的流式细胞仪平台用于胞浆内FVIII蛋白检测是一种可靠的方法,可通过揭示基因转移后细胞的功能状态来优化血友病A中的基因治疗方案。这表明需要确定安全的转导剂量窗口,以避免随后的细胞毒性。在这里,我们表明我们的流式细胞仪平台用于胞浆内FVIII蛋白检测是一种可靠的方法,可通过揭示基因转移后细胞的功能状态来优化血友病A中的基因治疗方案。
更新日期:2019-11-15
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