Targeted Oncolytic Therapy for Hepatocellular Carcinoma with High Specificity and Efficiency

Introduction and hypothesis: Hepatocellular Carcinoma (HCC) represents a significant global health burden, characterized by high mortality rates and limited therapeutic options. Annually, over 500,000 new cases of HCC are diagnosed globally, with the overall five-year survival rate for advanced stages ranging from a mere 5-9%. This study proposes a novel oncolytic virus (OV) approach for HCC treatment, hypothesizing that a combination of alpha-fetoprotein (AFP) promoter/enhancer system for precise targeting of HCC cells while sparing normal liver tissue, Diphtheria Toxin A (DTA) for potent cell lysis, and shGPX4 to overcome treatment resistance through ferroptosis pathway. Methodology: An Adeno-Associated Virus 2 (AAV2) vector was engineered to contain an AFP promoter/enhancer-driven DTA gene and a short hairpin RNA targeting GPX4 (shGPX4). The virus was produced in HEK293T cells, purified, and its efficacy was tested on different HCC cell lines (HepG2, Hep3B and Huh7) and normal hepatocyte cell line (MIHA) at various multiplicities of infection (MOIs). Cell viability was assessed using CCK8 assays. Moreover, constructed AAV2’s function on anti-resistance is tested on combination treatment with different-dosage Cisplatin. Results: The AFP promoter/enhancer was specifically selected based on comprehensive analysis of published dataset, which demonstrated significantly high AFP expression in liver cancer, correlation with disease outcomes, survival outcomes and specific epigenetic regulation in HCC. Further in vitro experiments demonstrated the construct AAV2 oncolytic virus has remarkable specificity and therapeutic efficacy. Multiple analysis revealed significant cytotoxicity in high-AFP-expressing HCC cell lines (HepG2 and Hep3B), reducing cell viability to approximately 40% at maximum MOI, while showing minimal impact on normal hepatocytes (MIHA). Moreover, combination therapy studies with cisplatin showed significant synergistic effects, particularly in chemoresistant cells, attributed to the GPX4 suppression mechanism. Conclusion: Our novel OV construct shows promising results in selectively targeting and eliminating HCC cells while sparing normal liver tissue. This approach addresses the critical needs for specificity and overcoming treatment resistance in HCC therapy. Further in vivo studies are warranted to fully elucidate its efficacy and safety, potentially leading to more effective and targeted treatments for hepatocellular carcinoma.