Construction and Optimization of Herpes Simplex Virus Vectors for CentralNervous System Gene Delivery based on CRISPR/Cas9-mediated GenomeEditing

作者：Xia Cao, Xinwei Huang, Xiu‐Qing Li, Lijuan Yang, Pengfei Wang, Jingyuan Yan, Zuqing Nie, Yingzheng Gao, Zhiwei Li, Jie Wen · 发表于：Current Gene Therapy · 年份：2021 · DOI：10.2174/1566523219666210618154326 · 被引用次数：8 · 研究领域：Herpesvirus Infections and Treatments、Virus-based gene therapy research、RNA regulation and disease

AIMS: We aim to define parameters that affect the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. BACKGROUND: Engineered, attenuated Herpes Simplex Virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and longterm transgene expression of attenuated HSV-1 vectors have not been fully understood. OBJECTIVE: The study aimed at using CRISPR-Cas9 system to construct attenuated HSV-1 vectors and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. METHODS: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and construct two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in mouse hippocampus gene transduction model. RESULTS: The in vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing ...