Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an “off-the-shelf” therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR-Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared with the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR-Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.

具有失活供体 T 细胞受体 (TCR) 表达的同种异体 CD19 特异性嵌合抗原受体 (CAR) T 细胞可用作淋巴恶性肿瘤的“现成”治疗方式，从而为自体、患者治疗提供了一种有吸引力的替代方案。衍生的 T 细胞。目前的 T 细胞工程方法主要依赖于病毒载体的使用。在这里，我们优化并验证了一个基于睡美人(SB) 转座子的非病毒基因修饰平台，该转座子带有小环，可表达 CD19-28z.CAR 和 CRISPR-Cas9 核糖核颗粒以灭活同种异体 TCR。以最小的细胞毒性实现了有效的 TCR 基因破坏，并实现了稳健且稳定的 CD19-28z.CAR 表达。CAR T 细胞对具有抗肿瘤活性的 CD19+ 肿瘤细胞有反应，可诱导 NALM6 荷瘤小鼠的肿瘤完全缓解，同时显着降低 TCR 同种异体反应性和 GvHD 发展。单一 CAR 信号传导在 TCR 破坏的 CAR T 细胞和对照 CAR T 细胞中诱导相似的 T 细胞信号传导特征。相比之下，在双重 CAR/TCR 信号传导过程中，与对照 CAR T 细胞相比，TCR 破坏抑制了 T 细胞信号传导/蛋白质磷酸化。这种非病毒 SB 转座子-CRISPR-Cas9 组合策略可作为生成下一代 CD19 特异性 CAR T 的替代方案，同时降低 GvHD 风险并缓解病毒载体固有的潜在制造限制。