Introduction: Lung adenocarcinoma (LUAD), the predominant subtype of non–small cell lung cancer (NSCLC), is frequently driven by pathogenic TP53 mutations, which promote tumor progression and therapeutic resistance. Restoration of wild-type p53 function through precise, transient genome editing represents a transformative therapeutic opportunity. However, safe and efficient in vivo delivery of gene-editing machinery to tumor cells remains a major challenge due to systemic toxicity, limited targeting, and potential off-target effects.
Methods: Here, we report the development of a PEGylated reduced-graphene-oxide (PEG-rGO) nanocarrier system engineered for targeted delivery of Cas9/ABE8e ribonucleoprotein (RNP) complexes to correct recurrent TP53 missense mutations in LUAD. The nanocarrier was functionalized with an RGD tumor-homing peptide and pH-responsive endosomal escape moieties to achieve selective tumor accumulation and efficient cytosolic release. Editing efficiency and specificity were assessed in patient-derived LUAD organoids via amplicon sequencing, while therapeutic efficacy was validated in orthotopic xenograft mouse models. Safety and biodistribution were evaluated using biochemical profiling, histopathology, and CIRCLE-seq/whole-genome sequencing analyses.
Results: In LUAD organoids (n = 7), PEG-rGO-mediated delivery achieved a median on-target TP53 correction of 59% (IQR, 52–66%), with minimal bystander editing (<0.15%) and no significant off-target events across 15 candidate loci. Corrected cells exhibited reactivation of the p53 pathway (CDKN1A/p21 ↑4.8×, BAX ↑3.2×, cleaved caspase-3 ↑), resulting in approximately 42% reduction in viability (Annexin V assay). In xenografted mice (n = 10 per group), systemic administration of PEG-rGO-RNP reduced tumor volume by 54% at day 28 and extended median survival from 31 to 47 days (p = 0.004). Tumor-to-liver accumulation ratio reached ≈2.6, with no significant alterations in ALT, AST, BUN, or hematologic parameters. Genome-wide specificity analysis confirmed ≤0.03% off-target editing. Multi-omics integration indicated reactivation of the canonical p53 → BAX → PUMA apoptotic cascade as the dominant antitumor mechanism.
Conclusion: This study establishes PEG-rGO–mediated RNP base editing as a robust, non-viral nanoplatform for precise correction of pathogenic TP53 alleles in lung adenocarcinoma. The results highlight its translational potential as a safe and effective strategy to restore tumor suppressor activity and overcome therapeutic resistance in p53-mutant cancers.