意大利圣拉斐尔科学研究所Eugenio Montini研究组从细胞游离DNA 中检测载体整合位点。这一研究成果于2021年6月17日发表在国际顶尖学术期刊《自然-医学》杂志上。

在这项研究中，他们开发了液体活检整合位点测序技术，或 LiBIS-seq，这是一种聚合酶链反应技术，经过优化，可从由多个组织中的死亡细胞释放到血液中的细胞游离DNA来定量检索载体整合位点。这种方法能够对接受造血干细胞基因疗法 (GT)  的患者体内肝脏定向 GT 和克隆追踪进行纵向监测，提高他们对实体组织中转基因细胞的克隆组成和更新的理解，并且与仅基于循环血细胞的传统分析相反，能够更早地检测到在外周组织中异常扩增的载体标记克隆。

据悉，GT作为一种治疗其他不治之症的方法迅速引起了人们的兴趣，市场上已经有几种 GT 产品，还有更多产品进入了选定适应症的临床试验。基于载体整合的克隆跟踪技术能够监测接受 GT 的患者血液中工程细胞的命运，并允许评估这些程序的安全性和有效性。然而，由于可以测试的细胞数量有限，而且在不进行侵入性活检的情况下研究驻留在外周器官的细胞是不切实际的，这种方法只能提供转基因细胞的克隆库和动力学的部分快照，并作为一种安全读数降低了预测能力。

附：英文原文

Title: Retrieval of vector integration sites from cell-free DNA

Author: Daniela Cesana, Andrea Calabria, Laura Rudilosso, Pierangela Gallina, Fabrizio Benedicenti, Giulio Spinozzi, Giulia Schiroli, Alessandra Magnani, Serena Acquati, Francesca Fumagalli, Valeria Calbi, Maximilian Witzel, Frederic D. Bushman, Alessio Cantore, Pietro Genovese, Christoph Klein, Alain Fischer, Marina Cavazzana, Emmanuelle Six, Alessandro Aiuti, Luigi Naldini, Eugenio Montini

Issue&Volume: 2021-06-17

Abstract: Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues. A new approach called liquid biopsy integration site sequencing enables monitoring of genetically modified cells in solid tissues of patients receiving gene therapy.

DOI: 10.1038/s41591-021-01389-4

Source: https://www.nature.com/articles/s41591-021-01389-4