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

Abstract

Coxsackievirus B3 (CVB3) has potential as a new oncolytic agent for the treatment of cancer but can induce severe pancreatitis. Here, we inserted target sequences of the microRNA miR‐375 (miR‐375TS) into the 5′ terminus of the polyprotein encoding sequence or into the 3′UTR of the CVB3 strain rCVB3.1 to prevent viral replication in the pancreas. In pancreatic EndoC‐βH1 cells expressing miR‐375 endogenously, replication of the 5′‐miR‐375TS virus and that of the 3′‐miR‐375TS virus was reduced by 4 × 103‐fold and 3.9 × 104‐fold, respectively, compared to the parental rCVB3.1. In colorectal carcinoma cells, replication and cytotoxicity of both viruses were slightly reduced compared to rCVB3.1, but less pronounced for the 3′‐miR‐375TS virus. Thus, CVB3 with miR‐375TS in the 3′UTR of the viral genome may be suitable to avoid pancreatic toxicity.

Coxsackievirus B3—Its Potential as an Oncolytic Virus

“…Nevertheless, several studies recently demonstrated that miR-TS can also be inserted into the 3′-UTR of CVB3 without affecting virus propagation. This was achieved by placing the miR-TS immediately downstream of the stop codon of the CVB3 polyprotein [74,103,[107][108][109]. This approach apparently prevents the destruction of critical secondary structures within the 3′-UTR of the virus.…”

“…This involved the insertion of miR-TS in a forward orientation to target the plus-strand genome or in a reverse orientation to target the minus-strand replication intermediate of CVB3. All CVB3 containing miR-TS in their plus-strand genome were sensitive to the corresponding miRs [74,103,105,[107][108][109]137]. The miR-targeting of the viral minus-strand is particularly attractive, as CVB3 generates only few minus-strand RNA intermediates during genome replication, which function as template for generating lots of plus-strand RNA genomes [141].…”

“…In vitro data have shown an impressive reduction in miR-TS-bearing CVB3 replication of up to 10 5 -fold in cells endogenously expressing their cognate miRs, which was corroborated in vivo. Even after the application of high doses of virus and the use of highly virulent virus strains, a complete to nearly complete abrogation of CVB3 replication and organ toxicity in tissues expressing corresponding miRs was observed in several studies [74,103,105,[107][108][109]. However, it should be mentioned that the degree of attenuation seemed to be affected by several factors, such as the absolute level of miR expression in normal tissues [107], the miR-TS copy number [103] and the route of viral application [105].…”

“…It has also been shown that miR-TS can be placed into the 5′-terminus of the CVB3 polyprotein encoding sequence. However, virus propagation in target cells, as well as inhibition of virus replication and cytotoxicity in cells expressing corresponding miRs were about 10-fold lower compared to CVB3 containing the miR-TS in the 3′-UTR [ 109 ]. Similar observations were made when comparing CVB3 with miR-TS in the 5′-UTR and 3′-UTR of the viral genome.…”

“…The miR-targeting of the viral minus-strand is particularly attractive, as CVB3 generates only few minus-strand RNA intermediates during genome replication, which function as template for generating lots of plus-strand RNA genomes [ 141 ]. However, targeting the minus-strand by miRs failed to inhibit viral replication [ 109 ] or merely showed a very low level reduction in viral replication compared to targeting the positive-strand genome [ 105 ]. The reason for the failure has been suggested to be the inaccessibility of the CVB3 minus-strand in the viral replication complexes to the RNA interference machinery [ 142 ].…”