科研级嵌合抗原受体慢病毒浓缩方法对比研究
Comparisons of Labtoratory-Grade Lentivirus Concentration Methods for Chimeric Antigen Receptor T Cell Preparation
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摘要: 慢病毒载体是基因治疗的重要基因转导工具, 在嵌合抗原受体(Chimeric Antigen Receptor, CAR)T 细胞治疗血液肿瘤中发挥着极其重要的作用。制备高质量 CAR-T 细胞的先决条件是获得高滴度的慢病毒, 而高滴度的实现离不开浓缩步骤。其中, 超速离心(超离)和超滤是两种潜在的可选浓缩方法。为探究超离和超滤两种浓缩方法在制备科研级 CAR-T 时的效率和对病毒的活力影响, 该研究选择 GFP 和 CD19 CAR(CAR19)-mCherry 两种质粒包装病毒, 并对两种方法产生的病毒滴度和回收率进行比较。结果显示, 超滤产生的慢病毒滴度最高达到 1011 TU/mL, 显著高于超离法产生的慢病毒滴度(P<0.05), 同时超滤的回收效率也更高。该结果可为科研级 CAR 慢病毒的浓缩方法提供直接的理论参考依据, 有助于 CAR-T 基础研究的顺利开展。Abstract: Lentiviral vector is an important gene transduction tool for gene therapy and plays an a crucial role in the treatment of hematologic tumors by chimeric antigen receptor (CAR) T cells. The prerequisite for the preparation of high quality CAR-T cells is to obtain lentivirus with high titer by concentration, and ultracentrifugation and ultrafiltration are two main methods for virus enrichment. In order to determine the efficiency of ultracentrifugation and ultrafiltration for virus concentration, and their effect on virus activity in the preparation of research-grade CAR-T, two lentivirus plasmids (GFP and CD19 CAR(CAR19)-mCherry) were selected to package lentivirus, then titer and recovery rate were compared. The results showed that the titer of virus produced by ultrafiltration reached 1011 TU/mL, which was significantly higher than that by ultracentrifugation (P<0.05), and the higher recovery efficiency was also obtained by ultrafiltration. These results can provide direct theoretical reference for the lentivirus concentration method to support the preparation of laboratory-grade CAR-T, and are helpful for the rapid development of CAR-T basic research.