Mengovirus, a member of thePicornaviridaefamily, has a broad cell tropism and can cause encephalitis and myocarditis in multiple mammalian species. Attenuation has been achieved by shortening the polycytidine tract in the 5' noncoding region (NCR). A poly(C)-truncated strain of mengovirus, vMC24, resulted in significant tumor regression in immunocompetent BALB/c mice bearing syngeneic MPC-11 plasmacytomas, but the associated toxicities were unacceptable. To enhance its safety profile, microRNA target sequences complementary to miR-124 or miR-125 (enriched in nervous tissue), miR-133 and miR-208 (enriched in cardiac tissue), or miR-142 (control; enriched in hematopoietic tissues) were inserted into the vMC24NCRs. The microRNA-detargeted viruses showed reduced replication and cell killing specifically in cells expressing the cognate microRNAs, but certain insertions additionally were associated with nonspecific suppression of viral fitnessin vivo. In vivotoxicity testing confirmed that miR-124 targets within the 5' NCR suppressed virus replication in the central nervous system while miR-133 and miR-208 targets in the 3' NCR suppressed viral replication in cardiac tissue. A dual-detargeted virus named vMC24-NC, with miR-124 targets in the 5' NCR and miR-133 plus miR-208 targets in the 3' NCR, showed the suppression of replication in both nervous and cardiac tissues but retained full oncolytic potency when administered by intratumoral (10(6)50% tissue culture infectious doses [TCID50]) or intravenous (10(7)to 10(8)TCID50) injection into BALB/c mice bearing MPC-11 plasmacytomas. Overall survival of vMC24-NC-treated tumor-bearing mice was significantly improved compared to that of nontreated mice. MicroRNA-detargeted mengoviruses offer a promising oncolytic virotherapy platform that merits further development for clinical translation. IMPORTANCE: The clinical potential of oncolytic virotherapy for cancer treatment has been well demonstrated, justifying the continued development of novel oncolytic viruses with enhanced potency. Here, we introduce mengovirus as a novel oncolytic agent. Mengovirus is appealing as an oncolytic virotherapy platform because of its small size, simple genome structure, rapid replication cycle, and broad cell/species tropism. However, mengovirus can cause encephalomyelitis and myocarditis. It can be partially attenuated by shortening the poly(C) tract in the 5' NCR but remains capable of damaging cardiac and nervous tissue. Here, we further enhanced the safety profile of a poly(C)-truncated mengovirus by incorporating muscle- and neuron-specific microRNA target sequences into the viral genome. This dual-detargeted virus has reduced pathogenesis but retained potent oncolytic activity. Our data show that microRNA targeting can be used to further increase the safety of an attenuated mengovirus, providing a basis for its development as an oncolytic platform.