National ''111'' Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, College of Bioengineering, Hubei University of Technology, Wuhan, 430068, PR China.
National ''111'' Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, College of Bioengineering, Hubei University of Technology, Wuhan, 430068, PR China; Wuhan Binhui Biopharmaceutical Co., Ltd. Wuhan, 430068, PR China.
Anal Biochem. 2024 Nov;694:115626. doi: 10.1016/j.ab.2024.115626. Epub 2024 Jul 18.
Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/μL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.
嵌合抗原受体 T 细胞（CAR-T）在癌症免疫治疗领域掀起了一场范式转变。逆转录病毒介导的基因转移将特定的 CAR 表达盒递送到 T 细胞中，跨越了癌症治疗的一系列基础研究和临床竞赛。然而，有必要设计一种精确和经过验证的定量方法，以适应不同的 CAR 构建体。在研究中，开发了一种 TaqMan 实时 qPCR 方法，利用靶向 ψ 基因序列的引物。该方法为评估整合 DNA 水平的逆转录病毒拷贝数提供了一种快速、敏感、可重复和准确的工具。重要的是，建立的 qPCR 与未转导的 T 细胞或组织没有交叉反应。表征 TaqMan 实时 PCR 动力学的回归方程为 y = -3.3841x + 41.402（R = 0.999），显示出 97.47%的扩增效率。值得注意的是，建立的 qPCR 方法的最小检测限为 43.1 拷贝/μL。此外，组内和组间差异均低于 4%，这表明该方法具有良好的可重复性。我们的体内和体外结果也支持其敏感性、特异性和稳定性。因此，该方法为研究人员提供了一种经济有效的工具，可用于体外和体内定量 CAR 拷贝数。
