Multiplexed nanoparticle delivery to increase CRISPR/Cas gene editing for enhanced cancer therapy

项目摘要 可编程CRISPR/Cas基因编辑系统具有巨大的癌症治疗潜力， 来精确地修复癌症相关基因。然而，将CRISPR递送至实体瘤以实现有效的治疗是不可能的。 癌症治疗仍然受到独特的僵硬和纤维化肿瘤微环境的限制， 纳米颗粒摄取。在这里，我们建议通过多重脂质直接靶向肿瘤组织力学， 纳米颗粒（LNP）方法，其涉及共递送粘着斑激酶（FAK）siRNA、Cas9 mRNA和 SgRNA（siFAK + CRISPR LNP）能够实现肿瘤递送并增强基因编辑功效。我们将利用 我们最近开发的非病毒选择性器官靶向（SORT）LNP平台， 核酸递送、蛋白质递送和静脉内（IV）施用后的基因组编辑。的 提出的方法涉及一种独特的siRNA介导的FAK基因沉默的组合，FAK是一种关键的调节剂， 和CRISPR介导的肿瘤相关基因的基因编辑（缺失），通过单一的多合一 纳米粒子方法。我们将进一步利用肝脏和肺SORT LNP来评估基因编辑作为一种治疗方法。 程序性死亡配体1（PD-L1）永久失活的策略，得到临床局限性的支持 抗PD-L1抗体atezolizumab治疗肝细胞癌（HCC）和非小细胞肺癌 （NSCLC）。在这项拨款提案中，我们的目标是（1）建立FAK敲低如何增强SORT LNP介导的 mRNA递送和CRISPR基因编辑，（2）优化肝脏和肺SORT LNP制剂， siRNA + Cas9 mRNA + sgRNA递送，和（3）评估肝和肺SORT siFAK的治疗功效 + 原位和基因工程小鼠癌症模型中的Cas9 mRNA + sgPD-L1递送。调节 用于增强肿瘤组织中遗传抑制的肿瘤细胞/ECM的机械性质提供了 利用CRISPR治疗癌症的创新策略。我们预计，这一总体方法可以进一步 在未来与其他类型的治疗剂协同作用。

Project Summary The programmable CRISPR/Cas gene editing system has great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR to solid tumors for efficient cancer therapy remains limited by the uniquely stiff and fibrotic tumor microenvironment that acts as a barrier to nanoparticle uptake. Here, we propose to directly target tumor tissue mechanics via a multiplexed lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA, and sgRNA (siFAK + CRISPR LNPs) to enable tumor delivery and enhance gene editing efficacy. We will leverage our recently developed non-viral Selective ORgan Targeting (SORT) LNP platform that enables tissue-specific nucleic acid delivery, protein delivery, and genome editing following intravenous (IV) administration. The proposed approach involves a unique combination of siRNA-mediated gene silencing of FAK, a key modulator of tumor ECM, and CRISPR-mediated gene editing (deletion) of tumor-related genes via a single all-in-one nanoparticle approach. We will further leverage Liver and Lung SORT LNPs for to evaluate gene editing as a strategy for permanent inactivation of programmed death-ligand 1 (PD-L1), supported by the clinical limitations of anti-PD-L1 antibody atezolizumab therapy in hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). In this grant proposal, we Aim to (1) establish how FAK knockdown enhances SORT LNP-mediated mRNA delivery and CRISPR gene editing, (2) optimize Liver and Lung SORT LNP formulations for multiplexed siRNA + Cas9 mRNA + sgRNA delivery, and (3) evaluate the therapeutic efficacy of Liver and Lung SORT siFAK + Cas9 mRNA + sgPD-L1 delivery in orthotopic and genetically engineered mouse models of cancer. Regulating the mechanical properties of tumor cells/ECM for enhancing the genetic suppression in tumor tissues provides an innovative strategy for treating cancer using CRISPR. We anticipate that this general approach could further synergize with additional types of therapeutics in the future.