CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

中文翻译：

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通过线性Doggybone DNA载体的piggyBac换位生成CAR T细胞需要转座子DNA侧翼区域。

使用病毒载体产生的CD19特异性嵌合抗原受体（CAR19）T细胞是治疗B细胞恶性肿瘤的有效方法，但费用昂贵。非病毒的piggyBac转座子系统为CAR19 T细胞的生产提供了一种简单而廉价的替代方法。到目前为止，piggyBac都是基于质粒的，有助于经济地在细菌中扩增载体。然而，扩增的质粒具有几种临床翻译所不希望的质量，包括细菌遗传成分，抗生素抗性基因以及纯化以去除内毒素的需求。狗骨头（dbDNA）是线性，共价封闭的，最小的DNA载体，可以在体外大规模酶促廉价地生产。重要的是，它们缺乏质粒的不良特性。我们使用结合了piggyBac的dbDNA生成CAR19 T细胞。最初，功能性转座酶的表达是明显的，但没有稳定的CAR表达。排除其他原因后，引入了位于dbDNA内转座子侧翼的其他随机DNA，从而促进了与使用质粒组分相当的稳定CAR表达。我们的发现表明掺入piggyBac的dbDNA可用于生成CAR T细胞，并表明需要在piggyBac转座子侧翼的DNA能够有效转座。dbDNA可以进一步降低转座子系统的成本并提高CAR T细胞生产的安全性。与使用质粒组分相比，可促进稳定的CAR表达。我们的发现表明掺入piggyBac的dbDNA可用于生成CAR T细胞，并表明需要在piggyBac转座子侧翼的DNA能够有效转座。dbDNA可以进一步降低转座子系统的成本并提高CAR T细胞生产的安全性。与使用质粒组分相比，可促进稳定的CAR表达。我们的发现表明掺入piggyBac的dbDNA可用于生成CAR T细胞，并表明需要在piggyBac转座子侧翼的DNA能够有效转座。dbDNA可以进一步降低转座子系统的成本并提高CAR T细胞生产的安全性。