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AAV-mediated combination gene therapy of inducible Caspase 9 and miR-199a-5p is therapeutic in hepatocellular carcinoma

Cancer Gene Therapy volume 31, pages 1796–1803 (2024)Cite this article

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Abstract

Advanced-stage hepatocellular carcinoma (HCC) remains an untreatable disease with an overall survival of less than one year. One of the critical molecular mediators contributing to increased resistance to therapy and relapse, is increased hypoxia-inducible factor 1α (HIF-1α) levels, leading to metastasis of tumor cells. Several microRNAs are known to be dysregulated and impact HIF-1α expression in HCC. An in silico analysis demonstrated that hsa-miR-199a-5p is downregulated at various stages of HCC and is known to repress HIF-1α expression. Based on this analysis, we developed a combinatorial suicide gene therapy by employing hepatotropic Adeno-associated virus-based vectors encoding an inducible caspase 9 (iCasp9) and miR-199a. The overexpression of miR-199a-5p alone significantly decreased ( ~ 2-fold vs. Mock treated cells, p < 0.05) HIF-1α mRNA levels, with a concomitant increase in cancer cell cytotoxicity in Huh7 cells in vitro and in xenograft models in vivo. To further enhance the efficacy of gene therapy, we evaluated the synergistic therapeutic effect of AAV8-miR-199a and AAV6-iCasp9 in a xenograft model of HCC. Our data revealed that mice receiving combination suicide gene therapy exhibited reduced expression of HIF-1α ( ~ 4-fold vs. Mock, p < 0.001), with a significant reduction in tumor growth when compared to mock-treated animals. These findings underscore the therapeutic potential of downregulating HIF-1α during suicide gene therapy for HCC.

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Fig. 1: Overexpression of miR-199a-5p downregulates HIF-1α and enhances cytotoxicity in vitro.
Fig. 2: Anti-tumorigenic effect of combination suicide gene therapy with AAV8MT-miR-199a vectors.
Fig. 3: AAV mediated miR-199a-5p overexpression downregulates HIF-1α in vivo.
Fig. 4: Cellular apoptosis in HCC tumors after combination suicide gene transfer.

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Data availability

Data generated in this study is available upon reasonable request to the corresponding author.

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Acknowledgements

We thank the central experimental animal facility, IIT-Kanpur, for housing the animals and cryosectioning facility. We acknowledge the assistance provided by Live Cell Imaging Lab, ACMS, and IIT Kanpur for confocal imaging. We also thank Dr. Saraswat Pathology, Kanpur, for preparing the paraffin sections and H&E staining of tumor samples. We thank Ms. Pratiksha Sarangi and Ms. Rupam Ashok Singh for technical help.

Funding

We acknowledge IIT Kanpur for providing intramural funding for this research work. S.P. and V.S. received study fellowships from the Human Resource Development Group, Council of Scientific & Industrial Research, and the Ministry of Human Resource Development, Government of India, respectively.

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Authors and Affiliations

  1. Laurus 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, Kanpur, 208016, India

    Subhajit Pathak, Vijayata Singh, Narendra Kumar G. & Giridhara R. Jayandharan

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  1. Subhajit Pathak
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  2. Vijayata Singh
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Contributions

SP, VS, and NK performed the experiments; SP and GRJ interpreted the data and wrote the manuscript; GRJ conceptualized and designed the study.

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Correspondence to Giridhara R. Jayandharan.

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Competing interests

The authors declare that IIT Kanpur has applied for patents on AAV vectors, and some of these have been licensed.

Ethical approval

All methods were performed in accordance with the relevant guidelines and regulations. Experiments related to recombinant DNA vectors and the production of AAV vectors have been approved by the Institute Bio-Safety Committee (IBSC), IIT Kanpur, India (Registration no. INDI14102019728). All animal-related experiments were conducted following approval from the Institute Animal Ethics Committee (IAEC), IIT Kanpur, India (Registration no. 810/GO/ReBi/S/2003/CPCSEA).

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Pathak, S., Singh, V., Kumar G., N. et al. AAV-mediated combination gene therapy of inducible Caspase 9 and miR-199a-5p is therapeutic in hepatocellular carcinoma. Cancer Gene Ther 31, 1796–1803 (2024). https://doi.org/10.1038/s41417-024-00844-x

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