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Research Article

基于CRISPR/ cas9基因编辑的中枢神经系统单纯疱疹病毒载体的构建与优化

卷 22, 期 1, 2022

发表于: 29 November, 2021

页: [66 - 77] 页: 12

弟呕挨: 10.2174/1566523219666210618154326

价格: $65

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摘要

目的:我们的目标是确定影响减毒HSV-1载体的安全性和长期转基因表达的参数，并优化表达盒，以实现在CNS中稳健和持续的表达。 背景:工程化、减毒的单纯疱疹病毒(HSV)载体是一种很有前途的将基因传递到外周和中枢神经系统的载体。病毒潜伏启动子(LAP)通常用于驱动外源基因的表达;然而，影响减毒HSV-1载体安全性和长期转基因表达的参数尚未完全了解。目的:利用CRISPR-Cas9系统构建HSV-1弱毒载体，检测转基因盒的构建和插入位点对转基因表达和载体安全性的影响。 方法:利用CRISPR-Cas9系统对HSV-1减毒株1716进行准确高效的编辑，构建LMR和LMRx两组重组病毒，分别在外显子1(LAP2)和LAP下游2.0 kb的内含子中插入不同的基因盒。在体外细胞系中比较了转基因基因的表达和病毒基因的转录动力学。在小鼠海马基因转导模型中进一步评估了各载体的报告基因表达和安全性。 结果:体外细胞系分析表明，基因表达盒的插入会破坏病毒基因的转录。小鼠海马转导分析表明，在2.0 kb内含子处插入完整的表达盒可以实现较长时间的基因表达。含有缺乏Poly (A)基因表达盒的重组子由于持续的病毒抗原表达和小胶质细胞激活而诱导了显著的神经元炎症。 结论:我们的结果表明，LAT转录本的完整性对于建立长期潜伏表达是不需要的。外源强启动子(如cBh启动子)置于LAT位点的外显子1或2.0 Kb内含子中，在潜伏期仍可保持活性，但其转录活性随着时间的延长而下降。与之前的研究一致，当基因盒位于外显子1的下游时，外源基因的表达持续时间更长，提示LAP2在潜伏期维持启动子活性中发挥作用。此外，LAT下游部分的过转录可诱导减毒载体的持续激活，提示LAT在维持病毒再激活潜能方面具有重要作用。

关键词: HSV-1载体，CRISPR-Cas9基因组编辑，潜伏期相关转录，海马基因转导，长期转基因表达，重组病毒。

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