Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust function, and improves survival in a Burkitt lymphoma xenograft model . Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

中文翻译：

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临床阶段高活性 TcBuster 转座酶的进化，作为过继细胞疗法的稳健非病毒生产平台

治疗人类疾病的细胞疗法，如嵌合抗原受体（CAR）T细胞和自然杀伤（NK）细胞在治疗血液恶性肿瘤方面显示出显着的临床疗效；然而，目前的方法主要利用病毒载体，但受到其载货量、成本高和临床试剂生产时间长的限制。通过 DNA 转座子工程传递遗传货物是一种更及时、更具成本效益的方法，但由于整合率较低而受到阻碍。在这里，我们报告了一种新型高活性 TcBuster (TcB-M) 转座酶的开发，该转座酶通过结构引导和进化方法进行设计，可在原代人类细胞中实现大型多顺反子 CAR 表达盒的高效整合。我们的 CAR-NK 和 CAR-T 细胞原理验证 TcB-M 工程显示出低整合载体拷贝数、安全的插入位点特征、强大的功能，并提高了伯基特淋巴瘤异种移植模型的存活率。总体而言，TcB-M 是一种多功能、安全、高效和开源的选择，可通过转位传递多顺反子大货物来快速制造和临床前测试初级人类免疫细胞疗法。