Development of an Optimized Promoter System for Exosomal and Naked AAV Vector-Based Suicide Gene Therapy in Hepatocellular Carcinoma

Abstract Suicide gene therapy is a promising strategy for the potential treatment of hepatocellular carcinoma (HCC). However, the lack of high transduction efficiency and targeted vectors in delivering the suicide genes to only the HCC cells is a major impediment. In the present study, we utilized an adeno-associated virus serotype 6 (AAV6) and its exosomal counterpart (exo-AAV) comprising of an inducible Caspase 9 (iCasp9) gene under the control of different promoter systems for targeting HCC cells. We employed a ubiquitous cytomegalovirus immediate early enhancer/chicken β actin promoter (CAG), a liver-specific promoter (LP1), and a baculoviral IAP repeat-containing protein 5 (BIRC5) promoter for liver and cancer cell-specific expression of iCasp9, respectively. We further evaluated these vectors in Huh7 cells for their ability to kill the target cells. BIRC5 and LP1 promoter-driven iCasp9 vectors demonstrated superior cytotoxicity when compared to CAG promoter-driven iCasp9 vectors. Further validation in a murine model of HCC demonstrated that the LP1-iCasp9 or Birc5-iCasp9-based AAV6 vectors contributed to tumor regression (∼2 fold) as effectively as the AAV6-CAG-iCasp9 vectors (∼1.9 fold). Similarly, exo-AAV6 vectors showed ∼2.1 to 2.8 fold superior in vivo tumor regression when compared to mock-treated animals. Our study has developed two novel promoters (LP1 or BIRC5) whose efficacy is comparable to a strong ubiquitous promoter in both AAV and exo-AAV systems. This expands the toolkit of AAV vectors for safe and effective treatment of HCC.