In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.
In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.
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DOI：
10.1038/mtna.2013.75
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发表时间：
2014-02-04
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急性感染后，单纯疱疹病毒（HSV）在感觉神经元中建立潜伏期，从中它可以重新激活并引起复发性疾病。现有的抗病毒治疗不影响潜伏病毒基因组;因此，它们不能防止治疗停止后的再激活。一种可能的治疗方法涉及通过稀切核酸内切酶在潜伏HSV基因组中引入DNA双链断裂，导致必需病毒基因的诱变。我们在体外HSV潜伏期模型中使用工程化归巢核酸内切酶（HE）HSV 1 m5测试了这种方法，该核酸内切酶识别HSV-1基因UL 19中编码病毒体蛋白VP 5的序列。3′-外切核酸酶Trex 2与HE的共表达使HE介导的突变频率增加了6倍。在用腺相关病毒（AAV）载体递送HSV 1 m5/Trex 2后，靶位点在潜伏HSV基因组中突变，没有可检测的细胞毒性。重要的是，HSV 1 m5/Trex 2暴露后，潜伏感染细胞在再活化后的HSV产生减少。在HSV 1 m5/Trex 2治疗前暴露于组蛋白脱乙酰酶抑制剂可使潜伏HSV基因组的诱变频率增加2 - 5倍，这表明染色质修饰可能是基因靶向方法的有用辅助手段。这些结果支持HE和其他核酸酶（ZFN、TALEN、CRISPR）用于治愈慢性病毒感染的持续开发。
Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.
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