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

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.

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

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 ß 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.

Inducible caspase 9-mediated suicide gene therapy using AAV6 vectors in a murine model of breast cancer.

Breast carcinoma has one of the highest incidence rates (11.7%), with significant clinical heterogeneity. Although conventional chemotherapy and surgical resection are the current standard of care, the resistance and recurrence, after these interventions, necessitate alternate therapeutic approaches. Cancer gene therapy for breast cancer with the suicide gene is an attractive option due to their directed delivery into the tumor. In this study, we have developed a novel treatment strategy against breast cancer with recombinant adeno-associated virus (AAV) serotype 6 vectors carrying a suicide gene, inducible Caspase 9 (iCasp9). Upon treatment with AAV6-iCasp9 vectors and the chemical inducer of dimerizer, AP20187, the viability of murine breast cancer cells (4T1) was significantly reduced to ∼40%-60% (mock control 100%). Following intratumoral delivery of AAV6-iCasp9 vectors in an orthotopic breast cancer mouse model, we observed a significant increase in iCasp9 transgene expression and a significant reduction in tumor growth rate. At the molecular level, immunohistochemical analysis demonstrated subsequent activation of the effector caspase 3 and cellular death. These data highlight the potential of AAV6-iCasp9-based suicide gene therapy for aggressive breast cancer in patients.

Role of Biomaterials in Cardiac Repair and Regeneration: Therapeutic Intervention for Myocardial Infarction.

Heart failure or myocardial infarction (MI) is one of the world's leading causes of death. Post MI, the heart can develop pathological conditions such as ischemia, inflammation, fibrosis, and left ventricular dysfunction. However, current surgical approaches are sufficient for enhancing myocardial perfusion but are unable to reverse the pathological changes. Tissue engineering and regenerative medicine approaches have shown promising effects in the repair and replacement of injured cardiomyocytes. Additionally, biomaterial scaffolds with or without stem cells are established to provide an effective environment for cardiac regeneration. Excipients loaded with growth factors, cytokines, oligonucleotides, and exosomes are found to help in such cardiac eventualities by promoting angiogenesis, cardiomyocyte proliferation, and reducing fibrosis, inflammation, and apoptosis. Injectable hydrogels, nanocarriers, cardiac patches, and vascular grafts are some excipients that can help the self-renewal in the damaged heart but are not understood well yet, in the context of used biomaterials. This review focuses on the use of various biomaterial-based approaches for the regeneration and repair of cardiac tissue postoccurrence of MI. It also discusses the outlines of cardiac remodeling and current therapeutic approaches after myocardial infarction, which are translationally important with respect to used biomaterials. It provides comprehensive details of the biomaterial-based regenerative approaches, which are currently the focus of the research for cardiac repair and regeneration and can provide a broad outline for further improvements.

Gene therapy for female infertility: A farfetched dream or reality?

A major cause of infertility in women is impaired ovulation or oogenesis. In this issue of Cell Reports Medicine, Kanatsu-Shinohara et al.1 demonstrate the potential of gene delivery with adeno-associated virus that can cross the blood-follicle barrier and restore oogenesis in congenitally infertile mice.