Quantitative Virus-Associated RNA Detection to Monitor Oncolytic Adenovirus Replication.

Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It is, however, difficult to obtain evidence for intratumoral oncolytic adenovirus replication if direct access to the tumor is not possible. Detection of systemic adenovirus DNA, which is sometimes used as a proxy, has limited value because it does not distinguish between the product of intratumoral replication and injected virus that did not replicate. Therefore, we investigated if detection of virus-associated RNA (VA RNA) by RT-qPCR on liquid biopsies could be used as an alternative. We found that VA RNA is expressed in adenovirus-infected cells in a replication-dependent manner and is secreted by these cells in association with extracellular vesicles. This allowed VA RNA detection in the peripheral blood of a preclinical in vivo model carrying adenovirus-injected human tumors and on liquid biopsies from a human clinical trial. Our results confirm that VA RNA detection in liquid biopsies can be used for minimally invasive assessment of oncolytic adenovirus replication in solid tumors in vivo.

Human Non-Small Cell Lung Cancer-Chicken Embryo Chorioallantoic Membrane Tumor Models for Experimental Cancer Treatments.

To promote the preclinical development of new treatments for non-small cell lung cancer (NSCLC), we established NSCLC xenograft tumor assays on the chorioallantoic membrane (CAM) of chicken embryos. Five NSCLC cell lines were compared for tumor take rate, tumor growth, and embryo survival. Two of these, A549 and H460 CAM tumors, were histologically characterized and tested for susceptibility to systemic chemotherapy and gene delivery using viral vectors. All cell lines were efficiently engrafted with minimal effect on embryo survival. The A549 cells formed slowly growing tumors, with a relatively uniform distribution of cancer cells and stroma cells, while the H460 cells formed large tumors containing mostly proliferating cancer cells in a bed of vascularized connective tissue. Tumor growth was inhibited via systemic treatment with Pemetrexed and Cisplatin, a chemotherapy combination that is often used to treat patients with advanced NSCLC. Lentiviral and adenoviral vectors expressing firefly luciferase transduced NSCLC tumors in vivo. The adenovirus vector yielded more than 100-fold higher luminescence intensities after a single administration than could be achieved with multiple lentiviral vector deliveries. The adenovirus vector also transduced CAM tissue and organs of developing embryos. Adenovirus delivery to tumors was 100-10,000-fold more efficient than to embryo organs. In conclusion, established human NSCLC-CAM tumor models provide convenient in vivo assays to rapidly evaluate new cancer therapies, particularly cancer gene therapies.

Evaluation of a Novel Oncolytic Adenovirus Silencing SYVN1.

Oncolytic adenoviruses are promising new anticancer agents. To realize their full anticancer potential, they are being engineered to express therapeutic payloads. Tumor suppressor p53 function contributes to oncolytic adenovirus activity. Many cancer cells carry an intact TP53 gene but express p53 inhibitors that compromise p53 function. Therefore, we hypothesized that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitors in selected cancer cells. To investigate this concept, we attenuated the expression of the established p53 inhibitor synoviolin (SYVN1) in A549 lung cancer cells by RNA interference. Silencing SYVN1 inhibited p53 degradation, thereby increasing p53 activity, and promoted adenovirus-induced A549 cell death. Based on these observations, we constructed a new oncolytic adenovirus that expresses a short hairpin RNA against SYVN1. This virus killed A549 cells more effectively in vitro and inhibited A549 xenograft tumor growth in vivo. Surprisingly, increased susceptibility to adenovirus-mediated cell killing by SYVN1 silencing was also observed in A549 TP53 knockout cells. Hence, while the mechanism of SYVN1-mediated inhibition of adenovirus replication is not fully understood, our results clearly show that RNA interference technology can be exploited to design more potent oncolytic adenoviruses.

Expression of Oncolytic Adenovirus-Encoded RNAi Molecules Is Most Effective in a pri-miRNA Precursor Format.

Oncolytic adenoviruses are being developed as new anti-cancer agents. Their efficacy can be improved by incorporating RNA interference (RNAi) molecules. RNAi molecules can be expressed in various precursor formats. The aim of this study was to determine the most effective format. To this end, we constructed three Δ24-type oncolytic adenoviruses, with human microRNA-1 (miR-1) expression cassettes in short hairpin RNA (shRNA), precursor microRNA (pre-miRNA), and primary miRNA (pri-miRNA) format, respectively. The viruses were compared for virus replication, mature miR-1 expression, and target gene silencing in cancer cells. Incorporation of the cassettes had only minor effects on virus replication. Mature miR-1 expression from the pri-miRNA format reached on average 100-fold higher levels than from the other two formats. This expression remained stable upon long-term virus propagation. Infection with the pri-miR-1-expressing virus silenced the validated miR-1 targets FOXP1 and MET. Drosha knockout almost completely abrogated mature miR-1 expression, confirming that processing of adenovirus-encoded pri-miR-1 was dependent on the host cell miRNA machinery. Using simple in vitro recombination cloning, a similar virus expressing miR-26b was made and shown to silence the validated miR-26b target PTGS2. We thus provide a platform for construction of oncolytic adenoviruses with high expression of RNAi molecules of choice.

Unleashing the Full Potential of Oncolytic Adenoviruses against Cancer by Applying RNA Interference: The Force Awakens.

Oncolytic virus therapy of cancer is an actively pursued field of research. Viruses that were once considered as pathogens threatening the wellbeing of humans and animals alike are with every passing decade more prominently regarded as vehicles for genetic and oncolytic therapies. Oncolytic viruses kill cancer cells, sparing healthy tissues, and provoke an anticancer immune response. Among these viruses, recombinant adenoviruses are particularly attractive agents for oncolytic immunotherapy of cancer. Different approaches are currently examined to maximize their therapeutic effect. Here, knowledge of virus⁻host interactions may lead the way. In this regard, viral and host microRNAs are of particular interest. In addition, cellular factors inhibiting viral replication or dampening immune responses are being discovered. Therefore, applying RNA interference is an attractive approach to strengthen the anticancer efficacy of oncolytic viruses gaining attention in recent years. RNA interference can be used to fortify the virus' cancer cell-killing and immune-stimulating properties and to suppress cellular pathways to cripple the tumor. In this review, we discuss different ways of how RNA interference may be utilized to increase the efficacy of oncolytic adenoviruses, to reveal their full potential.