High efficiency closed-system gene transfer using automated spinoculation.
High efficiency closed-system gene transfer using automated spinoculation.
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DOI：
10.1186/s12967-021-03126-4
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发表时间：
2021-11-24
影响因子：
7.4
通讯作者：
Highfill SL
中科院分区：
文献类型：
--
作者：
Remley VA;Jin J;Sarkar S;Moses L;Prochazkova M;Cai Y;Shao L;Liu H;Fuksenko T;Jin P;Stroncek DF;Highfill SL
基因转移是细胞治疗的重要工具。慢病毒载体最有效地转移到淋巴细胞或造血祖细胞使用spinoculation。为了实现符合cGMP（现行药品生产质量管理规范）的细胞疗法生产，我们开发并比较了使用细胞培养袋的封闭系统旋转接种方法和自动封闭系统旋转接种方法，以减少技术人员的动手时间并降低微生物污染的可能性。比较了Sepax离心接种、袋离心接种和静态袋转导（无离心接种）在单采收集的淋巴细胞中的慢病毒基因转移。将淋巴细胞转导一次并培养9天。测试的慢病毒载体编码CD 19/CD 22双特异性嵌合抗原受体（CAR）、FGFR 4-CAR或CD 22-CAR。通过测试袋旋转接种和静态转导来评价Sepax旋转接种时间，以优化Sepax旋转时间。然后使用Sepax spinoculation来测试不同CAR载体的转导。使用健康供体和患者样品评价了该过程的性能。使用针对NALM 6肿瘤细胞系的杀伤测定和细胞因子分泌分析对⑶ 19/22和⑶ 22 CAR T细胞进行功能评估。最后，检查转导的T细胞的基因表达，以确定是否存在可能由于自旋接种过程而发生的任何重大变化。棘球化过程导致基因转移的显著增强。使用1小时旋转时间的Sepax旋转接种显示出与袋旋转接种相当的转导效率，并且远高于静态袋转导方法（83.4%，72.8%，35.7%，n = 3）。三种不同方法的性能对于所有测试的慢病毒载体是一致的，并且当使用来自健康供体的起始细胞与患者样品时没有观察到显著差异。Sepax spinoculation不会影响CAR T细胞对抗肿瘤细胞的功能，因为这些细胞似乎同样可以杀死靶细胞。转种似乎也不影响基因表达模式，这是必要的赋予功能的细胞。封闭系统袋spinoculation导致更有效的淋巴细胞基因转移比标准袋转导无spinoculation。该方法对逆转录病毒和慢病毒载体在淋巴细胞中的基因转移都是有效的，并且可能是将基因转移到其他细胞类型（包括造血和髓系祖细胞）中的可行方法。Sepax离心接种进一步改进了该工艺，提供了一种自动化的封闭系统方法，显著缩短了操作时间，同时降低了培养袋撕裂和微生物污染的风险。在线版本包含补充材料，可通过10.1186/s12967-021-03126-4获得。
Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination. The online version contains supplementary material available at 10.1186/s12967-021-03126-4.
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DOI：
10.1097/cji.0b013e31818817c5
发表时间：
2008-11
期刊：
Journal of immunotherapy (Hagerstown, Md. : 1997)
影响因子：
--
作者：
Yang S;Rosenberg SA;Morgan RA
通讯作者：
Morgan RA
影响因子：
5.4
作者：
Guo, Jia;Wang, Weifeng;Wu, Yuntao
通讯作者：
Wu, Yuntao
影响因子：
20.3
作者：
Kochenderfer, James N.;Wilson, Wyndham H.;Rosenberg, Steven A.
通讯作者：
Rosenberg, Steven A.
携带抗Her2嵌合抗原受体基因的重组慢病毒生产方案的比较分析与优化
影响因子：
3.5
作者：
Yuan, Weihua;Chen, Jie;Li, Hongzhi
通讯作者：
Li, Hongzhi
影响因子：
4.5
作者：
Stroncek DF;Ren J;Lee DW;Tran M;Frodigh SE;Sabatino M;Khuu H;Merchant MS;Mackall CL
通讯作者：
Mackall CL