T细胞靶向稳定核酸脂质纳米粒用于体内产生白介素-6分泌功能缺失的嵌合抗原受体T细胞（CAR-T）研究

Chimeric antigen receptor T cells (CAR-T) are quite effective in the treatment of leukemia, but there are still two limitations: complicated in vitro preparation and cytokine release syndrome (CRS). This project aims to prepare a drug delivery system composed by CD19-CAR and interleukin-6 short hairpin RNA (IL-6 shRNA) integrated gene plasmid loaded stable nucleic acid lipid particles (SNALP) functionalized with CD3 single chain antibody (scFv) (CD19-CAR/IL-6 shRNA@SNALP-CD3 scFv). The nanoparticles can be targeted to T cells by CD3 scFv mediatation, and CD19-CAR/IL-6 shRNA integrated gene plasmid can enter cell nucleus and be stably transfected in T cells by nuclear localization signaling sequence mediation. Then CAR-T cells without the function of IL-6 secretion can be produced, which can specifically kill CD19 overexpressed leukemia tumor cells and decreased the IL-6 induced CRS, thereby improved the safety of CAR-T therapy. In this project, we solve the problems of CAR-T therapy by means of pharmaceutics. The CAR-T cells can be directly produced in vivo, avoiding the complicated in vitro preparation. This project provides a novel, convenient and safe implementation for CAR-T therapy, which can further promote the clinical application of CAR-T.

嵌合抗原受体T细胞（CAR-T）治疗白血病效果优异，但仍存在两大局限：复杂的体外制备流程和细胞因子释放综合征（CRS）。本项目拟构建一种CD3单链抗体（scFv）修饰的包载白介素-6短发夹RNA（IL-6 shRNA）和CD19-CAR组合基因质粒的稳定核酸脂质纳米粒（SNALP）递药系统（CD19-CAR/IL-6 shRNA@SNALP-CD3 scFv），以期在CD3 scFv介导下靶向至T细胞，所包载CD19-CAR/IL-6 shRNA组合基因质粒由核定位肽介导入核并稳定转染，产生IL-6分泌功能缺失的CD19-CAR-T，靶向杀伤CD19高表达白血病肿瘤细胞，同时降低IL-6引起的CRS，提高安全性。本项目利用药剂学手段解决CAR-T存在的问题，在体内直接产生CAR-T，避免复杂的体外制备流程，为CAR-T治疗提供一种新颖、便捷、安全的实施方案，可进一步推进CAR-T临床应用。

嵌合抗原受体T细胞（CAR-T）疗法在血液恶性肿瘤的治疗中取得了显著的成功。然而，其临床应用仍存在两个限制：传统CAR-T细胞复杂的体外制备流程和细胞因子释放综合征（CRS）。在本研究中，我们构建了一个CD3抗体修饰的脂质纳米粒系统，装载了含有白细胞介素6短发夹RNA（IL-6 shRNA）和CD19-CAR（AntiCD3-LNP/CAR 19+shIL6）组合基因质粒，使该LNP能通过CD3抗体的介导靶向并转染T细胞，在体内将T细胞转化为IL-6敲减的CAR-T细胞，从而杀伤CD19高表达的白血病肿瘤细胞，同时减少IL-6引起的CRS。体内实验表明，AntiCD3-LNP/CAR19+shIL6在体内能稳定转染T细胞，产生CAR-T杀伤肿瘤，并且CAR-T细胞可持续存在超过90天。该LNP显著延长了白血病模型小鼠的存活时间，达到与传统CAR-T相当的治疗效果。同时，给药小鼠的IL-6的表达被沉默，有助于减少CRS，提高CAR-T治疗的安全性。该方法避免了CAR-T复杂的体外制备流程，破除了限制CAR-T临床应用的关键瓶颈，极大提高了CAR-T技术的便捷性，有助于进一步促进CAR-T的临床应用。