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.