Abstract
Recognition sequences for microRNAs (miRs) that are down-regulated in tumor cells have recently been used to render lytic viruses tumor-specific. Since different tumor types down-regulate different miRs, this strategy requires virus customization to the target tumor. We have explored a feature that is shared by many tumor types, the up-regulation of miR-21, as a means to generate an oncolytic herpes simplex virus (HSV) that is applicable to a broad range of cancers. We assembled an expression construct for a dominant-negative (dn) form of the essential HSV replication factor UL9 and inserted tandem copies of the miR-21 recognition sequence (T21) in the 3' untranslated region. Bacterial Artificial Chromosome (BAC) recombineering was used to introduce the dnUL9 construct with or without T21 into the HSV genome. Virus was produced by transfection and replication was assessed in different tumor and control cell lines. Virus production was conditional on the presence of the T21 sequence. The dnUL9-T21 virus replicated efficiently in tumor cell lines, less efficiently in cells that contained reduced miR-21 activity, and not at all in the absence of miR-21. miR-21-sensitive expression of a dominant-negative inhibitor of HSV replication allows preferential destruction of tumor cells in vitro. This observation provides a basis for further development of a widely applicable oncolytic HSV.
Highlights
Oncolytic virus (OV) therapy is aimed at the selective destruction of cancer cells without harming healthy tissue [1]
Oncolytic viruses derived from Herpes Simplex Virus Type 1 have shown efficacy in preclinical models of several types of cancer and safety in Phase I human trials, but therapeutic outcomes have been disappointing [3
We inserted the same configuration (Tcon) and the miR-21 recognition sequence (T21) into the 3’UTR of a firefly luciferase expression plasmid and performed co-transfection experiments with a Pre-miR-21 or AntimiR-21 RNA on human U2OS and monkey Vero cells; human and monkey miR-21 are identical in sequence [48]
Summary
Oncolytic virus (OV) therapy is aimed at the selective destruction of cancer cells without harming healthy tissue [1]. OVs contain mutations that block lytic virus replication in normal cells but are complemented in cancer cells [2]. There is a need for new strategies to protect normal cells in the tumor environment while supporting undiminished virus replication in the tumor cells. Recent studies have demonstrated that lytic virus replication can be brought under the control of cellular microRNAs that are differentially expressed in normal and cancer cells [7,8,9]. Recognition sequences for microRNAs (miRs) that are down-regulated in tumor cells have recently been used to render lytic viruses tumor-specific. We have explored a feature that is shared by many tumor types, the up-regulation of miR-21, as a means to generate an oncolytic herpes simplex virus (HSV) that is applicable to a broad range of cancers.
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