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Development of an Optimized Promoter System for Exosomal and Naked AAV Vector-Based Suicide Gene Therapy in
Vijayata Singh1, Subhajit Pathak1, Narendra Kumar1
1Laurus Center for Gene Therapy, Department of Biological Sciences and Bioengineering and Mehta Family Center for Engineering in Medicine and Gangwal School of Medical Sciences and Technology, Indian Institute of Technology, Kanpur, Uttar Pradesh 208016, India.
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.
Insights
This study developed novel adeno-associated virus (AAV) vectors using specific promoters for hepatocellular carcinoma (HCC) gene therapy. These targeted vectors demonstrated effective tumor regression in preclinical models, offering a promising new treatment strategy.
Area of Science:
- Oncolytic Virotherapy
- Gene Therapy
- Molecular Oncology
Background:
- Hepatocellular carcinoma (HCC) treatment faces challenges due to inefficient targeted delivery of suicide genes.
- Adeno-associated virus (AAV) vectors and their exosomal forms (exo-AAV) show potential for targeted gene delivery.
- Developing specific promoters is crucial for enhancing the efficacy and safety of gene therapy vectors.
Purpose of the Study:
- To engineer AAV6 and exo-AAV6 vectors expressing an inducible Caspase 9 (iCasp9) suicide gene for HCC treatment.
- To evaluate the efficacy of different promoter systems (CAG, LP1, BIRC5) for liver and cancer cell-specific gene expression.
- To assess the in vitro and in vivo anti-tumor activity of these novel vectors in HCC models.
Main Methods:
- Construction of AAV6 and exo-AAV6 vectors encoding iCasp9 under CAG, LP1, or BIRC5 promoters.
- In vitro cytotoxicity assays using Huh7 HCC cells.
- In vivo tumor regression studies in a murine HCC model.
Main Results:
- LP1 and BIRC5 promoter-driven iCasp9 vectors exhibited superior cytotoxicity in vitro compared to the CAG promoter.
- Both AAV6 and exo-AAV6 vectors demonstrated significant tumor regression in vivo.
- Exo-AAV6 vectors showed enhanced tumor regression (2.1-2.8 fold) compared to mock-treated animals.
Conclusions:
- Novel LP1 and BIRC5 promoters achieve efficacy comparable to a strong ubiquitous promoter in AAV and exo-AAV systems.
- These engineered vectors expand the therapeutic toolkit for safe and effective HCC treatment.
- The study highlights the potential of targeted AAV-based gene therapy for hepatocellular carcinoma.
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