近日，美国耶鲁大学陈斯迪等研究人员合作发现，利用Cas13d对免疫抑制基因的多重抑制，可用于组合癌症免疫疗法。相关论文于2025年1月16日在线发表在《自然—生物技术》杂志上。

研究人员描述了一种通过基因沉默实现的多重普遍组合免疫疗法（MUCIG），该方法使用CRISPR–Cas13d沉默肿瘤微环境（TME）中的多个内源性免疫抑制基因，进而促进TME重塑并增强抗肿瘤免疫。

MUCIG载体通过腺相关病毒（AAV）递送，靶向四个基因（Cd274/Pdl1、Lgals9/Galectin9、Lgals3/Galectin3和Cd47；AAV-Cas13d-PGGC），在多个同源肿瘤模型中表现出显著的抗肿瘤疗效，并通过增加CD8⁺ T细胞浸润并减少中性粒细胞来重塑TME。

全转录组分析验证了四个靶基因的特异性敲低，并显示了有限的潜在脱靶或下游基因变化。AAV-Cas13d-PGGC的效果优于相应的shRNA治疗和单一基因敲低。

研究人员进一步通过采用高保真度Cas13d（hfCas13d）来优化MUCIG，结果显示其同样具有强大的基因沉默和体内抗肿瘤疗效，且没有体重下降或肝毒性。MUCIG代表了一种以可编程方式在体内沉默多个免疫基因的普适方法，能够在多种肿瘤类型中提供广泛的疗效。

据介绍，免疫抑制性TME的复杂性质需要多种药物联合使用，以实现最佳免疫疗法。

附：英文原文

Title: Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy

Author: Zhang, Feifei, Chow, Ryan D., He, Emily, Dong, Chuanpeng, Xin, Shan, Mirza, Daniyal, Feng, Yanzhi, Tian, Xiaolong, Verma, Nipun, Majety, Medha, Zhang, Yueqi, Wang, Guangchuan, Chen, Sidi

Issue&Volume: 2025-01-16

Abstract: The complex nature of the immunosuppressive tumor microenvironment (TME) requires multi-agent combinations for optimal immunotherapy. Here we describe multiplex universal combinatorial immunotherapy via gene silencing (MUCIG), which uses CRISPR–Cas13d to silence multiple endogenous immunosuppressive genes in the TME, promoting TME remodeling and enhancing antitumor immunity. MUCIG vectors targeting four genes delivered by adeno-associated virus (AAV) (Cd274/Pdl1, Lgals9/Galectin9, Lgals3/Galectin3 and Cd47; AAV-Cas13d-PGGC) demonstrate significant antitumor efficacy across multiple syngeneic tumor models, remodeling the TME by increasing CD8+ T-cell infiltration while reducing neutrophils. Whole transcriptome profiling validates the on-target knockdown of the four target genes and shows limited potential off-target or downstream gene alterations. AAV-Cas13d-PGGC outperforms corresponding shRNA treatments and individual gene knockdown. We further optimize MUCIG by employing high-fidelity Cas13d (hfCas13d), which similarly showed potent gene silencing and in vivo antitumor efficacy, without weight loss or liver toxicity. MUCIG represents a universal method to silence multiple immune genes in vivo in a programmable manner, offering broad efficacy across multiple tumor types.

DOI: 10.1038/s41587-024-02535-2

Source: https://www.nature.com/articles/s41587-024-02535-2