Marzulli M, Mazzacurati L, Zhang M, Goins W F, Hatley M E, Glorioso J C, Cohen J B
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh.
Department of Oncology, St. Jude Children's Research Hospital, USA.
J Gene Ther. 2018 Oct;3(1). doi: 10.13188/2381-3326.1000007. Epub 2018 Oct 18.
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 U9 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 dnU9 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 dnU9-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.
在肿瘤细胞中下调的微小RNA（miR）识别序列最近已被用于使裂解性病毒具有肿瘤特异性。由于不同的肿瘤类型下调不同的miR，这种策略需要针对目标肿瘤定制病毒。我们探索了许多肿瘤类型共有的一个特征，即miR-21的上调，以此作为产生适用于多种癌症的溶瘤性单纯疱疹病毒（HSV）的一种方法。
我们组装了一个用于必需的HSV复制因子U9的显性负性（dn）形式的表达构建体，并在3'非翻译区插入了miR-21识别序列（T21）的串联拷贝。利用细菌人工染色体（BAC）重组技术将带有或不带有T21的dnU9构建体引入HSV基因组。通过转染产生病毒，并在不同的肿瘤细胞系和对照细胞系中评估其复制情况。
病毒的产生取决于T21序列的存在。dnU9-T21病毒在肿瘤细胞系中高效复制，在miR-21活性降低的细胞中复制效率较低，而在没有miR-21的情况下则完全不复制。
HSV复制的显性负性抑制剂的miR-21敏感表达能够在体外优先破坏肿瘤细胞。这一观察结果为进一步开发广泛适用的溶瘤性HSV提供了基础。
