Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome.

Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21