Therapeutic Glypican-3 CRISPR Genome-Editing Using UltraLarge Porous Silica Nano-Depot for the Treatment of Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a key diagnostic marker and therapeutic target in hepatocellular carcinoma (HCC), interacting with Wnt and Hippo/YAP pathways related to cancer proliferation. Modulating GPC3 gene expression can induce liver cancer cell death by disrupting growth factor signaling, cell adhesion, and metabolic regulation. This study presents the development of a non-viral ultralarge porous CRISPR-Cas9 silica nano-depot to perform targeted GPC3 genome editing for the treatment of HCC. The synthesized ultralarge porous silica nano-depot (UPSND) encapsulates substantial CRISPR-Cas9-ribonucleoprotein complexes with a remarkable 98.3% loading efficiency. The UPSND-mediated GPC3 CRISPR-Cas9 therapy significantly suppresses cancer cell proliferation by modulating the Wnt and Hippo/YAP pathways. The efficiency of GPC3 gene deletion is observed to be 5.1-fold higher than that of commercial lipid-based GPC3 CRISPR-Cas9 in both human and murine genes, with minimal off-target effects. In vivo systemic administration of GPC3 Cas9-RNP@UPSND resulted in preferential accumulation within hepatic tissues in orthotopic HCC mouse models, leading to complete tumor eradication and enhancing tumor-infiltration. Furthermore, the GPC3 CRISPR-Cas9@UPSND treatment exhibits superior anti-proliferative efficacy in tumor-growth prevention compared to Codrituzumab, as evidenced by the analysis of Ki67 and GPC3 expression, along with serum GPC3 levels. These findings underscore the translational potential of the non-viral UPSND nanoplatform-based CRISPR GPC3 genome editing, offering a promising targeted therapeutic strategy for HCC treatment.