美国赋生元有限公司Hao Wu等研究人员合作，利用环状单链DNA介导的基因组整合进行高效非病毒免疫细胞工程。这一研究成果于2024年12月11日在线发表在国际学术期刊《自然—生物技术》上。

研究人员表示，腺相关病毒（AAV）作为同源重组（HDR）介导的基因组工程供体的使用受限于安全性问题、制造限制和包装容量的限制。非病毒靶向基因敲入主要依赖于双链DNA（dsDNA）和线性单链DNA（lssDNA）供体。已知dsDNA效率低且具有较高的细胞毒性，而lssDNA则在大规模制造中存在挑战。

研究人员开发了一种非病毒基因组写入催化剂（GATALYST）系统。该系统允许生产高达约20千碱基的环状单链DNA（cssDNA）作为供体模板，用于高效精准的转基因整合。

cssDNA供体在诱导多能干细胞（iPSC）中的敲入效率可高达70%，并且在多种临床相关的原代免疫细胞类型和多个基因组位点上显示出提高的效率，适用于各种核酸酶编辑系统的临床应用。

cssDNA在嵌合抗原受体（CAR）-T细胞和自然杀伤（NK）细胞中的，高精度和高效率、改进的安全性、负载灵活性以及可扩展制造能力，显示出其在未来基因组工程应用中的潜力。

附：英文原文

Title: Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration

Author: Xie, Keqiang, Starzyk, Jakob, Majumdar, Ishita, Wang, Jiao, Rincones, Katerina, Tran, Thao, Lee, Danna, Niemi, Sarah, Famiglietti, John, Suter, Bernhard, Shan, Richard, Wu, Hao

Issue&Volume: 2024-12-11

Abstract: The use of adeno-associated viruses (AAVs) as donors for homology-directed repair (HDR)-mediated genome engineering is limited by safety issues, manufacturing constraints and restricted packaging limits. Non-viral targeted genetic knock-ins rely primarily on double-stranded DNA (dsDNA) and linear single-stranded DNA (lssDNA) donors. dsDNA is known to have low efficiency and high cytotoxicity, while lssDNA is challenging for scaled manufacture. In this study, we developed a non-viral genome writing catalyst (GATALYST) system that allows production of circular single-stranded DNAs (cssDNAs) up to approximately 20 kilobases as donor templates for highly efficient precision transgene integration. cssDNA donors enable knock-in efficiency of up to 70% in induced pluripotent stem cells (iPSCs) and improved efficiency in multiple clinically relevant primary immune cell types and at multiple genomic loci implicated for clinical applications with various nuclease editor systems. The high precision and efficiency in chimeric antigen receptor (CAR)-T and natural killer (NK) cells, improved safety, payload flexibility and scalable manufacturability of cssDNA shows potential for future applications of genome engineering.

DOI: 10.1038/s41587-024-02504-9

Source: https://www.nature.com/articles/s41587-024-02504-9