In vivo inactivation of latent HSV by endonuclease-mediated mutagenesis

 描述（由申请人提供）：单纯疱疹病毒（HSV）感染仍然是一个常见、严重的问题，发病率很高。 HSV初次感染后形成潜伏期，目前的抗病毒治疗无法消除这种潜伏期，而潜伏病毒是病毒再激活和临床疾病复发的根源。尽管付出了很多努力，疫苗仍然难以找到。因此，需要一种能够治愈潜伏性HSV感染的新治疗方法。我们的假设是，设计师稀切核酸内切酶的出现作为定向基因组修饰的强大工具，提供了选择性靶向、切割和破坏活细胞内重要病毒 DNA 序列的独特能力。在这里，我们 建议使用归巢核酸内切酶（HE）通过必需病毒基因的定向诱变来禁用潜伏感染神经元中的HSV，从而消除病毒重新激活和复制的来源。该项目的目标是优化和评估我们使用 HSV 潜伏感染小鼠模型消除体内潜伏 HSV 感染的方法。此外，我们的结果将直接应用于治疗水痘带状疱疹病毒，这是另一种与单纯疱疹病毒一样在感觉神经元中建立潜伏期的α疱疹病毒。此外，生成的数据将与其他慢性或潜伏病毒感染（例如肝炎）的治疗方法的开发高度相关 B 病毒、HIV 或人乳头瘤病毒。在 SA1：评估潜伏 HSV 感染小鼠模型中 HSV DNA HE 诱变的体内功效中，我们将使用我们的潜伏 HSV 感染小鼠模型来测试 HE 诱变 HSV 并破坏其重新激活能力的能力。 I SA2：在潜伏性HSV感染的小鼠模型中评估和优化HSV DNA的HE诱变的体内安全性，我们将使用我们的潜伏性HSV感染的小鼠模型来评估和优化HE暴露的体内耐受性和安全性。该项目预计将证明我们旨在消除体内 HSV 发病机制的治疗方法的可行性，并为将这种新治疗方法推向临床所需的更大规模动物研究的发展提供关键信息。

 DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) infections remain a common, serious problem associated with significant morbidity. After primary infection HSV establishes latency, which is not eliminated by current antiviral therapy, and latent virus is the source for viral reactivation and the recurrence of clinical disease. Despite much effort, a vaccine remains elusive. Therefore, there is a need for a new therapeutic approach that would cure latent HSV infection. Our hypothesis is that the emergence of designer rare-cutting endonucleases as powerful tools for directed genome modification offers the unique ability to selectively target, cleave, and disrupt essential viral DNA sequences within living cells. Here, we propose to use homing endonucleases (HEs) to disable HSV in latently infected neurons by targeted mutagenesis of essential viral genes, eliminating the source of viral reactivation and replication. The goal of this project is to optimize and evaluate our approach to eliminate latent HSV infection in vivo using a murine model of HSV latent infection. Moreover, our results will be directly applicable in efforts to cure varicella zoster virus, another alphaherpesvirus that like HSV establishes latency in sensory neurons. Furthermore, the data generated will be highly relevant to the development of a cure of other chronic or latent viral infections such as hepatitis B virus, HIV, or human papillomavirus. In SA1: Evaluate the efficacy in vivo of HE mutagenesis of HSV DNA in a mouse model of latent HSV infection, we will use our mouse model of latent HSV infection to test the ability of HEs to mutagenize HSV and disrupt its ability to reactivate. I SA2: Evaluate and optimize the safety in vivo of HE mutagenesis of HSV DNA in a mouse model of latent HSV infection, we will use our mouse model of latent HSV infection to evaluate and optimize the in vivo tolerability and safety of HE exposure. This project is expected to demonstrate the feasibility of our therapeutic approach directed towards the elimination of HSV pathogenesis in vivo, and to provide critical information for the development of a larger scale animal study necessary to bring this new therapeutic approach to the clinic.