MicroRNA-Detargeted Mengovirus for Oncolytic Virotherapy

Abstract

Mengovirus, a member of the Picornaviridae family, 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 vMC24 NCRs. 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 fitness in vivo. In vivo toxicity 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 (106 50% tissue culture infectious doses [TCID50]) or intravenous (107 to 108 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.

MiR‐375‐mediated suppression of engineered coxsackievirus B3 in pancreatic cells

“…Despite the fact that our data indicate that insertion of miR‐TS into the viral genome per se slightly impairs virus replication and cytotoxicity, the site of miR‐TS insertion is an additional crucial factor affecting silencing of the virus by the corresponding miRs. The genomic structure of picornaviruses can be divided into a 5′‐UTR, the protein coding region, and a 3′UTR, and previously it was shown that CVB3 with miR‐TS in the 5′‐UTR and in the 3′UTR are susceptible to their corresponding miRs . On the other hand, it has also been shown that certain sections within the 5′‐UTR and 3′UTR do not tolerate miR‐TS insertion, most likely because insertion of miR‐TS disturbed higher‐order RNA structures of the viral genome .…”

“…In picornaviruses, the site of miR-TS insertion within the viral genome seems to be a further aspect critically influencing virus suppression. Previous published studies successfully inserted miR-TS within the 5 0 and 3 0 UTRs of the viral genome [14,23,27]. However, it has also been shown that certain sections within the 5 0 UTR and the 3 0 UTR do not tolerate miR-TS insertion [28].…”

Potential of oncolytic viruses in the treatment of multiple myeloma

“…Mengovirus (another member of Picornaviridae) has also been shown to infect and kill MM cells; however, in vivo studies with this virus demonstrated only modest efficacy along with relatively high toxicity. 134 Coxsackie viruses are positive-sense, single-stranded RNA viruses, which means their nucleic acid is directly infectious. In an interesting study, Hadac et al were able to demonstrate that injection of infectious A21 nucleic acid into solid MM plasmacytomas initiated an oncolytic infection with efficacy similar to that seen using intact virus.…”

MicroRNA-based Regulation of Picornavirus Tropism

“…The utility of this method for regulating viral tropism was originally demonstrated in lentiviral vectors to restrict transgene expression in specific tissues 14,15,16 . This technique has subsequently been applied to a vast array of replicating and non-replicating viral vectors for enhanced gene therapy as well as to improve the safety profiles of many oncolytic viruses by eliminating undesired toxicities in normal tissues 10,11,12,13,17 . It has also been utilized to generate safe and effective live-attenuated vaccines as well as to improve virus and vaccine manufacturing processes 18,19,20,21 .…”

“…This technique does not introduce new tropisms for the virus and therefore does not introduce any new safety concerns. Additionally, this method can be used to regulate multiple tropisms simultaneously by using target sequences for multiple different microRNAs enriched within different cell types 16,17,57,58,59,60,61 . This can all be accomplished without attenuating the virus in cells that do not express the corresponding microRNAs and therefore can provide a mechanism for improving the safety of therapeutic viruses with enhanced potency.…”