Endonuclease-mediated disruption of latent HSV as curative therapy

项目摘要 单纯疱疹病毒(HSV)感染仍然是一个常见、严重的问题，与显著 发病率。在初次感染后，单纯疱疹病毒会建立潜伏期，目前的抗病毒治疗无法消除这种潜伏期。 潜伏病毒是病毒重新激活和临床疾病复发的源头。尽管付出了很大的努力，一个 疫苗仍然难以捉摸。因此，需要一种新的治疗方法来治愈 潜伏的单纯疱疹病毒感染。我们一直在开发一种新的治疗策略来潜伏HSV感染，在这种策略中， 单纯疱疹病毒靶向内切酶诱导基本单纯疱疹病毒基因突变，使病毒基因组失效和 使病毒不能复制或从潜伏期重新激活。我们的假设是，最近的进展 在基因编辑技术和体内基因传递领域，提供了改善我们目前 抗病毒的方法和达到治疗效果。在这里，我们建议评估金黄色葡萄球菌 (SA)CRIPSR/Cas9和归巢内切酶(HES)在潜伏感染中使HSV失活的能力 通过靶向病毒基因组破坏神经元，从而消除动物体内病毒致病的根源 单纯疱疹病毒感染模型。该项目的目标是最大限度地提高我们的方法的有效性和安全性 用单纯疱疹病毒潜伏感染的小鼠模型消除体内潜伏的单纯疱疹病毒感染。我们的结果也将是 适用于治愈水痘带状疱疹病毒的另一种甲型疱疹病毒，与单纯疱疹病毒一样，在 感觉神经元。此外，生成的数据将与开发其他疾病的治疗方法高度相关 慢性或潜伏性病毒感染，如乙肝病毒、艾滋病毒或人类乳头瘤病毒。 在SA1：解决有效的体内HSV基因编辑的剩余障碍，我们将比较1)新的 我们目前的AAV血清型，通过不同的途径注射后用于转基因传递， 2)CRISPR/Cas9与HES的基因编辑能力。在SA2中：优化体内基因的疗效 编辑并确定对病毒致病机制的影响，我们将同时评估 靶向两个HSV位点提供了比靶向单个位点更好的疗效，并且基因的疗效 影响病毒致病机制的必要编辑。在SA3中：评估体内基因编辑在我们的 小鼠模型，我们将使用我们的潜伏感染HSV的小鼠模型来评估耐受性、安全性和 核酸酶暴露的遗传毒性。 这个项目有望证明我们的治疗方法的可行性 消除HSV在体内的致病机制，并为发展更大的HSV提供关键信息 将这一新的治疗方法引入临床所需的大规模动物研究。

Project Summary 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. 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 novel therapeutic approach that would cure latent HSV infection. We have been developing a new curative strategy to latent HSV infection, in which an HSV-targeted endonuclease induces mutagenesis of essential HSV genes, disabling viral genomes and rendering the virus incapable of replication or reactivation from latency. Our hypothesis is that recent advances in the field of gene-editing technologies and in vivo gene delivery offer the opportunity to improve our current anti-viral approach and reach therapeutic efficacy. Here, we propose to evaluate Staphylococcus aureus (Sa)CRIPSR/Cas9 and homing endonucleases (HEs) for their ability to disable HSV in latently infected neurons by targeted viral genome disruption, thus eliminating the source of viral pathogenesis in an animal model of HSV infection. The goal of this project is to maximize the efficacy and safety of our approach to eliminate latent HSV infection in vivo, using a murine model of HSV latent infection. Our results will also be 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 cures for other chronic or latent viral infections such as hepatitis B virus, HIV, or human papillomavirus. In SA1: Address the remaining barriers to effective in vivo HSV gene editing, we will compare 1) new AAV serotypes to our current AAV serotypes for transgene delivery after administration via different routes, and 2) the gene editing abilities of CRISPR/Cas9 vs. HEs. In SA2: Optimize the efficacy of in vivo gene editing and determine the impact on viral pathogenesis, we will evaluate whether the simultaneous targeting of two HSV sites provides superior efficacy over targeting a single site, and the efficacy of gene editing necessary to impact viral pathogenesis. In SA3: Evaluate the safety of in vivo gene editing in our mouse model, we will use our mouse model of latent HSV infection to evaluate tolerability, safety, and genotoxicity of nuclease 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.