Enhanced genomic stability of new miRNA-regulated oncolytic coxsackievirus B3

Genetic modification of coxsackievirus B3 (CVB3) by inserting target sequences (TS) of tumor-suppressive and/or organ-selective microRNAs (miRs) into viral genome can efficiently eliminate viral pathogenesis without significant impacts on its oncolytic activity. Nonetheless, reversion mutants (loss of miR-TS inserts) were identified as early as day 35 post-injection in ∼40% immunodeficient mice. To improve the stability, here we re-engineered CVB3 by (1) replacing the same length of viral genome at the non-coding region with TS of cardiac-selective miR-1/miR-133 and pancreas-enriched miR-216/miR-375 or (2) inserting the above miR-TS into the coding region (i.e., P1 region) of viral genome. Serial passaging of these newly established miR-CVB3s in cultured cells for 20 rounds demonstrated significantly improved stability compared with the first-generation miR-CVB3 with 5'UTR insertion of miR-TS. The safety and stability of these new miR-CVB3s was verified in immunocompetent mice. Moreover, we showed that these new viruses retained the ability to suppress lung tumor growth in a xenograft mouse model. Finally, we observed that miR-CVB3 with insertion in P1 region was more stable than miR-CVB3 with preserved length of the 5'UTR, whereas the latter displayed significantly higher oncolytic activity. Overall, we presented here valid strategies to enhance the genomic stability of miR-CVB3 for virotherapy.