miR-CAR-T细胞的制备及其功能研究

Adoptive transfer of T cells genetically engineered with chimeric antigen receptor (CAR) has shown great targeting and cytotoxic activity against malignance tumor cells and emerged as one of the most successful immunotherapeutic treatments for cancer patients. Our previous results have showed that anti-miR-301a can enhance cytotoxic activity of CAR-T cells against malignance tumor cells and inhibit cytokine storm. The data suggests that the more effective and safety of miR-CAR-T cells can be developed by engineered T cells combining with CAR and microRNA(miRNA). Therefore, we propose the following specific aims: Aim 1. To design miR-301a-CAR by integrated anti-miR-301a within lenti-virus and nonviral CAR vector, develop the miR-301a-CAR-T cells and evaluate the efficacy of miR-301a-CAR T cells to inhibit the growth of tumors. Aim 2. To screen and identify the miRNA which activate T cells and integrate it within CAR vector to develop the miR-CAR-T cells and further study its functions in vivo and in vitro. Aim 3. To screen and identify the miRNA which regulate cytokine expression and integrate it within CAR vector to develop the miR-CAR-T cells and evaluate its cytotoxic activity and safety in vivo and in vitro. Finally, this project would develop the more efficient and safety miR-CAR-T cells by genetically engineered with miRNA and CAR to immunotherapy for malignance cancer patients.

以嵌合抗原受体（CAR）修饰T细胞为基础的过继细胞免疫技术在恶性肿瘤免疫治疗中显示了很好的靶向性和杀伤活性。我们前期研究表明加入单个miR-301a阻遏物的CAR-T细胞可以增强其杀伤肿瘤活性和降低细胞因子风暴（如IL-6和TNFα等的分泌），结果提示我们:整合miRNA和CAR修饰技术，可以开发更有效和安全的新一代miR-CAR-T细胞。本项目进一步研究：1. 整合miR-301a阻遏物到表达CAR的病毒或非病毒载体，制备miR-301a-CAR-T细胞，进行功能和活性鉴定；2. T细胞激活miRNA的筛选、鉴定和CAR的整合，体内和体外研究其功能；3. 靶向细胞因子调节miRNA的筛选、鉴定和CAR的整合，研究其体内体外活性并进行安全性评估。本项目最终将整合miRNA和CAR修饰技术编辑T细胞，开发高效、安全的新一代miR-CAR-T细胞用于恶性肿瘤的细胞免疫治疗。

本项目首先筛选到了miR-153靶向调节IDO1表达，并且增强CAR-T细胞介导的肿瘤免疫治疗。我们结果发现：IDO1在结直肠肿瘤中高度表达，并且与患者存活率负相关。miR-153直接通过靶向其在结肠癌细胞中的 3'UTR来抑制 IDO1 的表达；然而，miR-153过表达不影响癌细胞的存活、凋亡和集落形成。当结肠癌细胞被 CAR-T细胞靶向时，肿瘤细胞内miR-153过表达显着增强体外T细胞杀伤并抑制异种移植肿瘤小鼠的生长。研究表明miR-153抑制结肠癌细胞中IDO1的表达，并且是一种增强CAR-T细胞免疫治疗的肿瘤抑制性miRNA。我们进一步研究了肿瘤微环境缺失miR-301a可以通过提升Runx3和招募CD8+T细胞有效减少肿瘤转移，而肿瘤细胞中的miR-301a敲低本身通过提高Runx3表达来抑制细胞迁移。加入单个miR-301a阻遏物的PD-L1-CAR-T细胞可以增强其杀伤肿瘤活性和降低细胞因子表达，如IL-6和TNFα等的分泌。我们研究了靶向PD-L1的CAR-T细胞机制，我们构建了靶向PD-L1的CAR- T细胞，并评估了它们在PD-L1高表达或低表达的NSCLC中的疗效。PD-L1-CAR-T细胞表现出针对PD-L1高NSCLC细胞和异种移植肿瘤的抗原特异性活化、细胞因子产生和细胞毒活性。 此外，加入亚治疗剂量的局部放疗提高了PD-L1-CAR-T细胞对PD-L1low NSCLC 细胞和肿瘤的疗效。 我们的研究结果表明，PD-L1-CAR-T 细胞代表了一种新的治疗策略，用于PD-L1 阳性NSCLC患者，特别是对那些易患HPD的患者。最后，我们开发了一种基于CRISPR腺嘌呤碱基编辑器（ABE）的基因敲除新方法i-Silence，有望后续联合CART细胞疗法。