67. Rational Design of MicroRNA-Retargeted Coxsackievirus A21 Infectious RNA for Cancer Therapy

Abstract

Coxsackievirus A21 (CVA21) is a positive-sense single stranded RNA virus. Intratumoral (IT) administration of CVA21 viral particles or in vitro transcribed RNA encoding the CVA21 genome to SCID mice bearing subcutaneous KAS6/1 human multiple myeloma xenografts results in tumor regression and viremia, but causes lethal myositis. We previously showed that inserting target sequences complementary to muscle-specific microRNAs (miRT), specifically miR-206 and miR-133, into the 3’ UTR of the viral genome inhibits viral replication in cells expressing the cognate miRNAs. IT administration of miRT-virus to KAS6/1 tumor bearing mice regressed tumors and ameliorated muscle toxicity. However, IT injection of RNA encoding the miRT-CVA21 genomes failed to induce tumor regression. In vitro transfected miRT-CVA21 RNA had delayed rescue kinetics and blind serial passaging was required to produce titratable virus. Therefore, the goal of this study was to optimize miRT positioning within the genomic construct to achieve retargeting without compromising virus rescue efficiency. We inserted two copies of each miRT in the variable domain (VD) of the 5’ UTR to make CVA21-T2-VD. We also truncated the VD ad replaced it with either one or two copies of each miRT generating CVA21-T1-ΔVD and CVA21-T2-ΔVD. Finally, we hypothesized that the original 3’ UTR insertion position deforms a stabilizing secondary RNA structure that incorporates part of the ORF and the 3’UTR thus hindering viral replication. To preserve the stability of this structure we duplicated the terminal sequences of the ORF and inserted either one or two copies of each miRT in between, forming CVA21-T1-TR and CVA21-T2-TR. Transfection of RNA encoding each of the recombinant genomes or the wildtype genome into H1-HeLa cells resulted in visible cytopathic effects at 48 hours. Viral titers produced at 12 hours post-transfection with CVA21, CVA21-T1-ΔVD, CVA21-T2-ΔVD, CVA21-T2-VD, CVA21-T1-TR and CVA21-T2-TR RNA were 1.6×105, 1.4×104, 4.0×104, 3.1×105, 9.2×104 and 3.7×104 TCID50/ml, respectively. The integrity of the miRT sequences and the absence of additional mutations were confirmed by sequencing. All five miRT-CVA21 viruses displayed replication kinetics similar to that of unmodified CVA21 during one-step growth curve analysis. miRNA targeting was analyzed by measuring the viability of H1-HeLa cells transfected with synthetic miR-206, miR-133 or both followed by viral infection. Cells transfected with muscle specific miRNAs resisted oncolysis by CVA21-T1-ΔVD, CVA21-T2-ΔVD, CVA21-T2-VD and CVA21-T1-TR to variable degrees but not by wildtype CVA21 or CVA21-T2-TR. Maximal protection was achieved in cells transfected with both miRNAs. Correspondingly, viral titers measured in supernatants of cells resisting oncolysis were lower than viral titers in supernatants of cells succumbing to it. Conclusion: In this study we have generated microRNA-retargeted CVA21 infectious RNA that results in the rescue of viral particles at a similar rate to unmodified CVA21 RNA. In conjunction with our previous studies, this data supports the use of microRNA-retargeted infectious RNA as a potential formulation for oncolytic virotherapy. Animal studies to test in vivo efficacy and selectivity of intratumorally administered RNA corresponding to the aforementioned CVA21 recombinants are currently underway.