在过去的十年中，用于治疗血液恶性肿瘤的嵌合抗原受体（CAR）-T细胞疗法经历了巨大的进展。然而，仍需解决一些关键限制，以进一步提高疗效、减轻毒性，以确保CAR-T细胞的持久性、到达肿瘤部位、抵抗恶性肿瘤微环境（TME）和限制毒性，通过释放能够将免疫抑制TME转化为有利于免疫排斥的因子以实现对TME的上下文释放，从而改善强大但潜在有毒的生物制品的产生。我们使用聚集的规律间隔短回文重复序列（CRISPR）激活（CRISPRa）系统创建了一种HER2-靶向CAR-T（RB-312），该系统通过条件转录其两个内源亚单位p35和p40来诱导IL-12异源二聚体的表达。该电路包括两个慢病毒构建体。第一个（HER2-TEV）表达抗人表皮生长因子受体2（HER2）CAR单链变量片段（scFv），带有CD28和CD3z共刺激结构域，链接到烟草切割病毒（TEV）蛋白酶和两个单导RNA（sgRNA），分别针对亚型互素（IL）-12A和IL12B转录起始位点（TSS）。第二个构建体（LdCV）编码激活T细胞的链接蛋白（LAT），通过TEV可切割序列（TCS）与核酸酶失活链球菌（dCas9）-VP64-p65-Rta（VPR）融合。CAR的激活将HER2-TEV与LdCV带至近距离接触，释放dCas9进行核定位。这种条件电路导致条件性和可逆诱导IL-12 / p70异源二聚体表达。在体外，RB-312与控制组（cRB-312）和常规HER2 CAR（convCAR）进行了比较。诱导性CRISPRa系统激活内源性IL-12表达，导致增强的次生干扰素（FN）-γ产生，细胞毒性和CAR-T增殖，以及体内持续时间延长，抑制HER2 + FaDu口咽癌细胞生长，与常规CAR-T细胞产品相比。周围循环中未检测到系统性IL-12。此外，与程序性死亡配体（PD-L1）阻断相结合显示出强大的协同作用。RB-312是第一个具有CRISPRa系统的临床相关产品，具有非基因编辑和可逆上调内源基因表达的功能，促进CAR-T细胞对HER2表达的肿瘤治疗的持久性和有效性。可逆且纳米级别的IL-12产生的自分泌作用限制了肿瘤外泄和系统性毒性的风险。©2023年作者（们）。

Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection.We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR).The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects.RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.© 2023. The Author(s).