Lin Huan-Ting, Okumura Takashi, Yatsuda Yukinori, Ito Satoru, Nakauchi Hiromitsu, Otsu Makoto
1 Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .
2 Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .
Hum Gene Ther Methods. 2016 Oct;27(5):197-208. doi: 10.1089/hgtb.2016.059.
Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer-probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.
通过整合型病毒载体将基因稳定转移到靶细胞群体中，在干细胞基因治疗（SCGT）中被广泛应用。准确估计载体拷贝数（VCN）变得越来越重要。然而，现有的估计方法，如实时光定量PCR，在实际应用中受到更多限制，特别是在临床试验期间，因为患者的样本材料有限。本研究展示了一种名为液滴数字PCR（ddPCR）的新兴技术在SCGT背景下估计VCN状态的应用。从一名患有X连锁慢性肉芽肿病的患者中获得的诱导多能干细胞（iPSC）被用作可克隆的靶细胞，用携带密码子优化的CYBB cDNA的α逆转录病毒载体进行转导。精确的引物-探针设计随后进行多重分析赋予了检测特异性。无需依赖参考标准曲线即可准确估计每细胞的VCN值。灵敏度高且检测动态范围广。通过观察转导的具有不同转基因拷贝数的iPSC克隆的一致、可重复和独特的VCN聚类模式，验证了检测的可靠性。综上所述，ddPCR的使用似乎为VCN估计提供了一种实用且强大的方法，具有广泛的临床和研究应用。
