Engineering Rapalog-Inducible Genetic Switches Based on Split-T7 Polymerase to Regulate Oncolytic Virus-Driven Production of Tumour-Localized IL-12 for Anti-Cancer Immunotherapy.

The approval of different cytokines as anti-neoplastic agents has been challenged by dose-limiting toxicities. Although reducing dose levels affords improved tolerability, efficacy is precluded at these suboptimal doses. Strategies combining cytokines with oncolytic viruses have proven to elicit potent survival benefits in vivo, despite promoting rapid clearance of the oncolytic virus itself. Herein, we developed an inducible expression system based on a Split-T7 RNA polymerase for oncolytic poxviruses to regulate the spatial and temporal expression of a beneficial transgene. This expression system utilizes approved anti-neoplastic rapamycin analogues for transgene induction. This treatment regimen thus offers a triple anti-tumour effect through the oncolytic virus, the induced transgene, and the pharmacologic inducer itself. More specifically, we designed our therapeutic transgene by fusing a tumour-targeting chlorotoxin (CLTX) peptide to interleukin-12 (IL-12), and demonstrated that the constructs were functional and cancer-selective. We next encoded this construct into the oncolytic vaccinia virus strain Copenhagen (VV-iIL-12mCLTX), and were able to demonstrate significantly improved survival in multiple syngeneic murine tumour models through both localized and systemic virus administration, in combination with rapalogs. In summary, our findings demonstrate that rapalog-inducible genetic switches based on Split-T7 polymerase allow for regulation of the oncolytic virus-driven production of tumour-localized IL-12 for improved anti-cancer immunotherapy.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Implications for SARS-CoV-2 Vaccine Design: Fusion of Spike Glycoprotein Transmembrane Domain to Receptor-Binding Domain Induces Trimerization.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presents an urgent need for an effective vaccine. Molecular characterization of SARS-CoV-2 is critical to the development of effective vaccine and therapeutic strategies. In the present study, we show that the fusion of the SARS-CoV-2 spike protein receptor-binding domain to its transmembrane domain is sufficient to mediate trimerization. Our findings may have implications for vaccine development and therapeutic drug design strategies targeting spike trimerization. As global efforts for developing SARS-CoV-2 vaccines are rapidly underway, we believe this observation is an important consideration for identifying crucial epitopes of SARS-CoV-2.