Kim Jaeryuk, Park Miyoung, Baek Gyungwon, Kim Joo-Il, Kwon Euna, Kang Byeong-Cheol, Kim Jong-Il, Kang Hyoung Jin
Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Republic of Korea.
Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Republic of Korea.
Mol Ther Oncolytics. 2023 May 16;30:1-13. doi: 10.1016/j.omto.2023.05.004. eCollection 2023 Sep 21.
Integration site (IS) analysis is essential in ensuring safety and efficacy of gene therapies when integrating vectors are used. Although clinical trials of gene therapy are rapidly increasing, current methods have limited use in clinical settings because of their lengthy protocols. Here, we describe a novel genome-wide IS analysis method, "detection of the integration sites in a time-efficient manner, quantifying clonal size using tagmentation sequencing" (DIStinct-seq). In DIStinct-seq, a bead-linked Tn5 transposome is used, allowing the sequencing library to be prepared within a single day. We validated the quantification performance of DIStinct-seq for measuring clonal size with clones of known IS. Using chimeric antigen receptor (CAR)-T cells, we revealed the characteristics of lentiviral IS. We then applied it to CAR-T cells collected at various times from tumor-engrafted mice, detecting 1,034-6,233 IS. Notably, we observed that the highly expanded clones had a higher integration frequency in the transcription units and vice versa in genomic safe harbors (GSH). Also, in GSH, persistent clones had more frequent IS. Together with these findings, the new IS analysis method will help to improve the safety and efficacy of gene therapies.
当使用整合型载体时，整合位点（IS）分析对于确保基因治疗的安全性和有效性至关重要。尽管基因治疗的临床试验正在迅速增加，但由于当前方法的方案冗长，在临床环境中的应用有限。在此，我们描述了一种新的全基因组IS分析方法，“高效检测整合位点，使用转座酶标签测序定量克隆大小”（DIStinct-seq）。在DIStinct-seq中，使用了与珠子相连的Tn5转座体，使得测序文库能够在一天内制备完成。我们用已知IS的克隆验证了DIStinct-seq测量克隆大小的定量性能。使用嵌合抗原受体（CAR）-T细胞，我们揭示了慢病毒IS的特征。然后，我们将其应用于从荷瘤小鼠在不同时间收集的CAR-T细胞，检测到1034 - 6233个IS。值得注意的是，我们观察到高度扩增的克隆在转录单元中的整合频率更高，反之在基因组安全港（GSH）中则相反。此外，在GSH中，持续存在的克隆具有更频繁的IS。连同这些发现，新的IS分析方法将有助于提高基因治疗的安全性和有效性。
