Whelan JT, Singaravelu R, Wang F, Pelin A, Tamming LA, Pugliese G, Martin NT, Crupi MJF, Petryk J, Austin B, He X, Marius R, Duong J, Jones C, Fekete EEF, Alluqmani N, Chen A, Boulton S, Huh MS, Tang MY, Taha Z, Scut E, Diallo JS, Azad T, Lichty BD, Ilkow CS, and Bell JC
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., Competing Interests: JB and BL are scientific co-founders of Turnstone Biologics, which develops oncolytic vaccinia viruses. JD, MH, MT, and AP have worked for Turnstone Biologics. JB and BL are shareholders in Turnstone Biologics. JB, CI, RS, FW and BL are co-inventors on a patent regarding the expression of bacterial cyclases and their therapeutic utility in mammals. RS is currently employed by the Public Health Agency of Canada (PHAC). His contribution to the study described herein was conducted prior to his employment at PHAC. The work was not undertaken under the auspices of PHAC and does not represent the views of PHAC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Whelan, Singaravelu, Wang, Pelin, Tamming, Pugliese, Martin, Crupi, Petryk, Austin, He, Marius, Duong, Jones, Fekete, Alluqmani, Chen, Boulton, Huh, Tang, Taha, Scut, Diallo, Azad, Lichty, Ilkow and Bell.)