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由AAV介导的基因组编辑受R循环的形成的影响

Francesco Puzzo^1,2, Magdalena P Crossley³, Aranyak Goswami^1,2

¹Department of Genetics, Stanford University, Stanford, CA.

bioRxiv : the preprint server for biology

|May 20, 2024

概括

基因组R环,在高度转录的基因中发现的结构,促进了腺相关病毒载体 (AAV) 的集成. 调节R循环可以提高基因治疗的安全性,并预测非目标整合部位.

科学领域:

分子生物学分子生物学
遗传学是一个遗传学.
基因治疗基因治疗

背景情况:

复合腺相关病毒载体 (rAAV) 广泛用于体内基因治疗,因为它们能够刺激同源复合 (AAV-HR).
然而,将rAAV整合到基因组中可能会导致非目标突变,这引发了安全问题.

研究的目的:

研究基因组R循环在rAAV整合和同源重组中的作用.
为了比较转化细胞系 (HEPA1-6) 和完整的肝脏组织中的R循环形成.
探索调制R环的潜力,以提高基因编辑的安全性和有效性.

主要方法:

在小鼠HEPA1-6肝瘤细胞和整个小鼠肝脏中进行了DNA-RNA免疫沉降测序 (DRIP-seq).
实验涉及R环的遗传和药理上调.
专辑蛋白基因被用作基因编辑研究的模型基因 in vitro 和 in vivo.

主要成果:

在具有上调R环的小鼠中观察到增强的AAV-HR.
阿尔伯明基因的R-循环易受3'末端被AAV-HR有效编辑,而R-循环缺陷区域显示没有可检测的整合.
之前报告的目标外的rAAV集成站点和R环丰富的基因组区域之间发现了正相关性.

结论:

在高度转录的基因中,高水平的R循环促进了rAAV载体基因组的整合.
调节R环可能提供一种策略,以提高rAAV介导的基因组编辑的安全性和有效性.
了解R循环动态可以帮助预测易受rAAV插入性突变发生的基因组区域.

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