基于标记的分析揭示了CAR-T细胞的克隆行为与慢载体整合位点相关。

IF 5.3 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
Jaeryuk Kim, Miyoung Park, Gyungwon Baek, Joo-Il Kim, Euna Kwon, Byeong-Cheol Kang, Jong-Il Kim, Hyoung Jin Kang
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引用次数: 0

摘要

当使用整合载体时,整合位点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,但由于其冗长的协议,目前的方法在临床环境中的应用有限。在这里,我们描述了一种新的全基因组IS分析方法,“以高效的方式检测整合位点,使用标记测序定量克隆大小”(DIStinct-seq)。在DIStinct-seq中,使用了头部连接的Tn5转座体,允许在一天内准备好测序文库。我们用已知IS的克隆验证了DIStinct-seq测定克隆大小的定量性能。利用体外嵌合抗原受体(CAR)-T细胞,我们揭示了慢病毒IS的特征。然后,我们将其应用于不同时间从肿瘤移植小鼠收集的CAR-T细胞,检测到1,034-6,233个IS。值得注意的是,我们观察到高度扩增的克隆在转录单位中具有更高的整合频率,反之亦然。此外,在GSH中,持久克隆更频繁地发生IS。结合这些发现,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。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites.

Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites.

Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites.

Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites.

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 ex vivo 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.

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来源期刊
Molecular Therapy Oncolytics
Molecular Therapy Oncolytics Medicine-Oncology
CiteScore
10.90
自引率
3.50%
发文量
152
审稿时长
6 weeks
期刊介绍: Molecular Therapy — Oncolytics is an international, online-only, open access journal focusing on the development and clinical testing of viral, cellular, and other biological therapies targeting cancer.
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