Department of Molecular Virology, Pasture Institute of Iran, Tehran, Iran.
Faculty of Life Science and Biotechnology, Department of Cell and Molecular Biology, Shahid Beheshti University, Tehran, Iran.
PLoS One. 2023 Oct 20;18(10):e0286231. doi: 10.1371/journal.pone.0286231. eCollection 2023.
Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.
溶瘤病毒 (OVs) 已成为一种新型癌症治疗方法，它能选择性地靶向并杀死癌细胞，同时不影响正常细胞。其中，经过工程改造的单纯疱疹病毒 1 型 (HSV-1) 已被提议作为癌症的潜在治疗方法，并已进入 III 期临床试验。先前的研究表明，OV 治疗与 p53 基因治疗的联合设计可增强 OV 的抗癌活性。在这里，利用 CRISPR/Cas9 编辑方法对 ICP34.5 缺失的 HSV-1 的 UL39 基因进行操作，插入 EGFP-p53 表达盒，以增强肿瘤选择性和肿瘤毒性能力。通过利用鸡胚绒毛尿囊膜 (CAM) 作为补充膜来支持具有基因缺陷的病毒生长，从感染的鸡胚 CAM 中分离出 ΔUL39/Δγ34.5/HSV1-p53 突变体。与体外的亲本 Δγ34.5/HSV-1 相比，ΔUL39/Δγ34.5/HSV1-p53 感染细胞的表型特征表明，HSV-1-P53 在来自不同起源、具有不同 p53 表达率的各种细胞系中具有细胞溶解能力。总的来说，这里呈现的数据说明了利用 CAM 模型作为一种有前途的策略来分离重组病毒的可行性，例如 CRISPR/Cas9 介导的 HSV-1-P53 突变体，由于外源性 p53 诱导的细胞死亡率增加，在细胞系中病毒复制减少。
