Abstract 320: CRISPR-mediated glypican-3 genome editing via ultra-large porous silica nanoparticles in hepatocellular carcinoma therapy

Abstract Purpose: We aim to develop non-viral nanocarriers to deliver ribonucleoproteins (RNP) targeting Glypican-3 (GPC3) and investigate the biological and immunological therapeutic effects from CRISPR-Cas9 GPC3 gene editing in hepatocellular carcinoma (HCC). Methods: Ultra-large porous silica nanoparticles (ULSN) were synthesized following established methods, with morphology confirmed via electron microscopy. ULSN-loaded Cas9-RNP was characterized using Zetasizer NS90, and RNP content measured by NanoDrop and BCA assays (ThermoFisher). In vitro experiments were performed on HepG2 (HB-8065, ATCC) and Hepa1-6 (CRL-1830, ATCC) cells. Gene deletion was assessed via RT-PCR, deep sequencing (Illumina MiniSeq), and cell viability. Western blotting analyzed key signaling pathways. In vivo GPC3 genome editing was evaluated in Hepa1-6 orthotopic HCC models (C57BL6, male, 6-7 weeks) using T7E1 assay and T cell infiltration analysis. GPC3 targeting therapeutic effects were compared with Codrituzumab (anti-GPC3) treatment in HepG2-bearing HCC models (Balb/c, male, 6-7 weeks) by measuring tumor volume, immunohistology (H&E, Ki67, and GPC3), serum GPC3, and T cell populations from tumor-draining lymph nodes using flow cytometry (BD). Results: ULSN exhibited a size of 300 nm, pore size of 22.86 nm, and surface area of 525.9 m2/g as determined by TEM and BET analyses. Its ultra-large pores allowed a 27% loading capacity for Cas9-RNP, with a loading efficiency of 98% for both sgRNA and Cas9, compared to 48% and 52% for Lipofectamine (commercial control), respectively. In HepG2 cells, GPC3 deletion showed 67% reduction in RT-PCR, 15% indel frequency (vs. 3% for Lipofectamine), and reduced cell viability (55% vs. 94%). To analyze the molecular effects of GPC3 gene, Wnt and Hippo/YAP signaling were examined. Western blotting revealed downregulation of Wnt signaling (lower level of Wnt, p-LRP, and β-catenin), upregulation of Hippo signaling through increased LATS phosphorylation, and decreased YAP phosphorylation, potentially inhibiting tumor cell proliferation. In orthotopic HCC models, ULSN showed targeted accumulation in liver tissues, successfully achieving GPC3 deletion with complete tumor regression 14 days post-procedure. Tumor cell death led to enhanced CD4+ and CD8+ T cell infiltration, alongside a decrease in regulatory T cells. Additionally, ULSN-mediated GPC3 gene editing outperformed Codrituzumab (GPC3 blockade) by reducing tumor establishment, Ki67 and GPC3 expression levels in tumor cells. Conclusions: ULSN presents strong potential as a non-viral delivery system for CRISPR-Cas9 RNP targeting GPC3 in HCC, offering improved gene delivery efficiency over lipid-based nanomaterials. This approach not only enhances tumor growth suppression, but also promotes T cell infiltration, ensuring effective and lasting therapeutic outcomes through GPC3 deletion. Citation Format: Sanghee Lee, Sian Lee, Hyojin Lee, Seongchan Kim, Dong-Hyun Kim. CRISPR-mediated glypican-3 genome editing via ultra-large porous silica nanoparticles in hepatocellular carcinoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 320.