MicroRNA-based Regulation of Picornavirus Tropism

Abstract

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

MicroRNA-detargeting proves more effective than leader gene deletion for improving safety of oncolytic Mengovirus in a nude mouse model

“…Picornavirus toxicity can be subdued through miRNA-mediated detargeting to prevent viral replication in off-target tissues, which is a well-studied technique. 1 , 13 , 50 , 51 , 52 This technique has worked very effectively for improving the safety profile of Mengovirus. The dual-detargeted Mengovirus, vMC 24 NC, showed potent therapeutic efficacy in a syngeneic plasmacytoma MPC-11 model without significant neuro- and cardiotoxicity.…”

“…Owing to this, miRNA-detargeting has become an effective method for selectively controlling the tissue tropism and toxicity of a variety of RNA and DNA viruses. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 Our laboratory has previously demonstrated that the miRNA-detargeting strategy can be used to moderate the neuro- and cardiotoxicities associated with a poly(C) truncated oncolytic Mengovirus, vMC 24 , while retaining the virus’ ability to replicate in a wide range of murine and human cancer cells. 12 This virus, vMC 24 NC, generated by inserting neuron- and cardiac/muscle-specific miRNA targets into the 5′ and 3′ non-coding regions (NCRs) of the viral genome, showed excellent therapeutic efficacy against a syngeneic murine plasmacytoma (MPC-11) model without significant virus-mediated toxicity.…”

“…Despite the success of miRNA-detargeting in controlling pathogenesis of a variety of oncolytic viruses, particularly picornaviruses, development of escape mutants and possible reversion to a virulent phenotype remains a concern with little to no clinical data. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 Although virus escape from miRNA-mediated inhibition may be less of a concern in immunocompetent hosts, as the virus can be cleared prior to evolution, the safety of these viruses in immunocompromised hosts needs to be thoroughly analyzed.…”

Oncolytic virus delivery: from nano-pharmacodynamics to enhanced oncolytic effect

“…Micro-RNA (miRNA) is evolving as a regulator of vector tropism, 120 , 121 which is in contrast to the initial descriptions of RNA viruses where vector tropism could not be controlled through transcriptional targeting. This was evident in the case of coxsackievirus type A21, which causes off-target severe, often fatal myositis.…”

The long-lasting enigma of polycytidine (polyC) tract

“…Because of their positive-sense RNA genomes, oncolytic picornaviruses are especially suitable for another form of post-viral entry replication control, which hijacks the miRNA gene expression control network to regulate viral permissivity. miRNA-targeting elements inserted within the viral genome target picornaviruses for degradation in healthy tissues expressing the cognate miRNA, while replication occurs unencumbered in neoplastic tissues where the cognate miRNA is not expressed [ 116 ]. This approach was shown to reliably eliminate fatal CAV21-associated myotoxicity in OV mouse models, both in the infectious RNA and viral particle formulation [ 117 – 119 ].…”