Immune, Progenitor and Cell Therapeutics Laboratory, Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, Mayo Clinic, Rochester, Minnesota, USA.
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, Rochester, MN, USA.
Cytotherapy. 2021 May;23(5):452-458. doi: 10.1016/j.jcyt.2020.12.012. Epub 2021 Mar 11.
Viral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct.
Assay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations.
The limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10-10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations).
DDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.
病毒载体常用于将嵌合抗原受体 (CAR) 构建体引入细胞治疗产品中，以治疗人类疾病。它们在基因传递方面效率很高，并整合到宿主基因组中进行后续复制，但如果最终产品中仍存在复制型慢病毒 (RCL)，则存在风险。理想的 CAR T 细胞产品应含有足够的整合病毒物质且无 RCL。目前的产品检测方法包括细胞为基础的检测方法，其具有缓慢的周转时间，以及快速定量聚合酶链反应 (PCR) 检测方法，这些方法存在结果变异性高的问题。作者描述了一种用于检测水疱性口炎病毒 G 糖蛋白包膜序列的液滴数字 PCR (ddPCR) 方法的开发，该序列是病毒组装所必需的，并且该方法还可用于测量 CAR 构建体的整合复制反应元件。
检测方法的验证包括在低至高浓度范围内的精密度、线性、灵敏度、特异性和重现性。
检测限为 10 拷贝/μL，而阴性样本显示 <1.3 拷贝/μL。在报告范围内（10-10 000 拷贝/μL），批内和批间精密度变异系数 <25%。通过将已知拷贝数与测量的拷贝数进行比较，验证了准确性和线性（R >0.9985，斜率~0.9）。最后，连续测量显示出非常好的长期重现性（在最初建立的 ± 两个标准差内，超过 95%的重复结果）。
ddPCR 具有出色的重现性、线性、特异性和灵敏度，可快速检测 RCL 并确保患者产品的安全性。该技术也可能适用于细胞产品制造过程中快速检测其他目标，包括纯度、效力和无菌性检测。
