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有效的非病毒免疫细胞工程使用循环单链DNA介导的基因组集成

Keqiang Xie¹, Jakob Starzyk², Ishita Majumdar²

¹Full Circles Therapeutics, Cambridge, MA, USA. keqiang.xie@fullcirclestx.com.

Nature biotechnology

|December 11, 2024

概括

这项研究引入了一种新的非病毒基因组写作催化剂 (GATALYST) 系统,使用圆形单链DNA (cssDNA) 捐赠者进行精确的基因组工程. 这种方法实现了高的敲进效率和提高了安全性,克服了病毒载体和其他非病毒方法的局限性.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
生物技术是生物技术.

背景情况:

基因相关病毒 (AAVs) 面临同质导向修复 (HDR) 介导的基因组工程的局限性,原因是安全,制造和包装的限制.
目前使用双链DNA (dsDNA) 的非病毒方法表现出低效率和高细胞毒性,而线性单链DNA (lssDNA) 则提出了制造可扩展性的挑战.

研究的目的:

开发一种新的非病毒系统,用于高效和精确的基因组工程,使用圆形单链DNA (cssDNA) 作为捐赠者模板.
克服与AAVs和现有的非病毒DNA捐赠者用于基因整合的局限性.

主要方法:

基因组编写催化剂 (GATALYST) 系统的开发,用于生产高达大约20千基的cssDNA捐赠者.
测试cssDNA供体对诱导多能干细胞 (iPSC) 和原发性免疫细胞 (包括CAR-T和NK细胞) 中转基因集成的效率.
在临床相关的基因组位置使用各种核酶编辑器系统评估cssDNA性能.

主要成果:

在使用cssDNA捐赠者的iPSC中实现了高达70%的内置效率.
在多种主要免疫细胞类型和各种基因组位点中证明了提高效率.
在仿真抗原受体 (CAR) -T和自然杀手 (NK) 细胞中展示了高精度和效率.

结论:

cssDNA捐赠系统为精密基因组工程提供了一个有前途的非病毒替代方案,超越了当前病毒和非病毒方法的局限性.
GATALYST系统展示了增强的安全性,有效载荷灵活性和可扩展的制造能力,这表明未来临床基因组工程应用的巨大潜力.

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