尽管我们对 1 型单纯疱疹病毒 (HSV-1) 生物学的了解已大大增强，但制定消除潜伏感染个体中的 HSV-1 的治疗策略仍然是一个公共卫生问题。目前用于治疗 HSV-1 并发症的抗病毒药物不具有特异性，并且不能解决潜伏感染。我们最近开发了一种基于 CRISPR-Cas9 的基因编辑平台，专门针对 HSV-1 基因组。在这项研究中，我们进一步使用 2D Vero 细胞培养物和 3D 人类诱导多能干细胞衍生的脑类器官 (CO) 模型来评估我们针对病毒 ICP0 或 ICP27 基因的编辑构建体的有效性。我们发现，在 Vero 细胞中用 AAV2-CRISPR-Cas9 载体靶向 ICP0 或 ICP27 基因可显着抑制 HSV-1 复制。此外，我们用 HSV-1 有效地感染了 CO，表征了病毒复制动力学，并建立了病毒潜伏期模型。最后，我们发现靶向 ICP0 或 ICP27 的 AAV2-CRISPR-Cas9 载体显着减少了潜伏感染 HSV-1 的 CO 中的病毒反弹。总之，我们的结果表明 HSV-1 的 CRISPR-Cas9 基因编辑是消除潜伏病毒库并治疗 HSV-1 相关并发症的有效治疗方法。© 2024 作者。

Although our understanding of herpes simplex virus type 1 (HSV-1) biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR-Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D human induced pluripotent stem cell-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 genes. We found that targeting the ICP0 or ICP27 genes with AAV2-CRISPR-Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR-Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR-Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1-associated complications.© 2024 The Author(s).