Tommasi Anna, Cappabianca Dan, Bugel Madison, Gimse Kirstan, Lund-Peterson Karl, Shrestha Hum, Arutyunov Denis, Williams James A, Police Seshidhar Reddy, Indurthi Venkata, Davis Sage Z, Murtaza Muhammed, Capitini Christian M, Saha Krishanu
Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA.
Mol Ther Methods Clin Dev. 2025 Feb 18;33(1):101437. doi: 10.1016/j.omtm.2025.101437. eCollection 2025 Mar 13.
CRISPR-Cas9 ribonucleoproteins (RNPs) combined with a nucleic acid template encoding a chimeric antigen receptor (CAR) transgene can edit human cells to produce CAR T cells with precise CAR insertion at a single locus. However, many human cells have adverse innate immune responses to foreign nucleic acids, particularly circular double-stranded DNA (dsDNA). Here, we introduce Cleaved, LInearized with Protein Template (Cas9-CLIPT), a circular plasmid containing a single target sequence for the Cas9 RNP, such that during manufacturing, Cas9-RNP binds and cleaves the plasmid to linearize the dsDNA . Cas9-RNP remains bound to the linearized template and is delivered to cells to promote precise knock-in via homology-directed repair with Cas9-CLIPT. Cas9-CLIPT Nanoplasmids generate up to 1.7-fold higher rates of precise knock-in relative to linearized dsDNA, reaching efficiencies up to 60% with non-homologous end joining inhibition. Cas9-CLIPT-manufactured GD2 -CAR T cells are potent against GD2 neuroblastoma cells and exhibit an enriched stem cell memory phenotype. On several electroporation instruments and approaching clinically relevant yields, we successfully manufactured -CAR T cells using Cas9-CLIPT plasmids containing large (2-6 kb) transgenes. Cas9-CLIPT strategies have the potential to simplify donor template production and integrate large transgenes, allowing for more efficient nonviral manufacturing of multifunctional, genome-edited immune cell therapies.
CRISPR-Cas9核糖核蛋白（RNP）与编码嵌合抗原受体（CAR）转基因的核酸模板相结合，可以编辑人类细胞，以在单个位点精确插入CAR从而产生CAR T细胞。然而，许多人类细胞对外源核酸，尤其是环状双链DNA（dsDNA）有不良的先天免疫反应。在此，我们引入了用蛋白质模板切割、线性化的（Cas9-CLIPT），这是一种含有单个Cas9 RNP靶序列的环状质粒，使得在制备过程中，Cas9-RNP结合并切割该质粒以使dsDNA线性化。Cas9-RNP仍然与线性化模板结合，并被递送至细胞，以通过与Cas9-CLIPT的同源定向修复促进精确敲入。相对于线性化dsDNA，Cas9-CLIPT纳米质粒产生的精确敲入率高达1.7倍，在抑制非同源末端连接的情况下效率可达60%。用Cas9-CLIPT制备的GD2 -CAR T细胞对GD2神经母细胞瘤细胞具有强大作用，并表现出富集的干细胞记忆表型。在几种电穿孔仪器上并接近临床相关产量时，我们使用含有大（2-6 kb）转基因的Cas9-CLIPT质粒成功制备了-CAR T细胞。Cas9-CLIPT策略有潜力简化供体模板生产并整合大的转基因，从而实现更高效的多功能、基因组编辑免疫细胞疗法的非病毒制备。
