Igarashi Yuka, Uchiyama Toru, Minegishi Tomoko, Takahashi Sirirat, Watanabe Nobuyuki, Kawai Toshinao, Yamada Masafumi, Ariga Tadashi, Onodera Masafumi
Department of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, Japan.
Department of Pediatrics, Hokkaido University Graduate School of Medicine, Hokkaido 060-8638, Japan.
Mol Ther Methods Clin Dev. 2017 May 25;6:8-16. doi: 10.1016/j.omtm.2017.05.005. eCollection 2017 Sep 15.
Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34 cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.
原发性免疫缺陷的干细胞基因治疗（SCGT）的临床改善取决于基因校正细胞的植入水平，因此追踪每个造血谱系中的转基因对于评估SCGT的疗效极为重要。我们建立了一种基于单细胞的液滴数字PCR（sc-ddPCR）方法，该方法包括将单个细胞封装到每个液滴中，然后用针对转基因的引物和探针进行乳液PCR。液滴中的荧光信号表明基因组中存在携带靶基因的单个细胞，该系统可以在基因组水平上清楚地确定整个群体中转基因阳性细胞的比例。我们使用sc-ddPCR分析了两名接受SCGT治疗的重症联合免疫缺陷（SCID）患者中载体转导细胞的植入情况。转导细胞的充分植入仅限于外周血（PB）中的T细胞谱系，并且一小部分CD34细胞在骨髓中表现出载体整合，这表明PB中的转基因阳性细胞可能已从一小部分干细胞或谱系受限的前体细胞分化而来。sc-ddPCR是一种简化且强大的工具，可用于详细评估接受SCGT治疗的患者中转基因阳性细胞的分布。
