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针对过度活跃的整合酶的定向进化,用于特异性插入转基因的特定位置

Brian E Hew¹, Sabranth Gupta¹, Ryuei Sato¹

¹Department of Cell and Molecular Biology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96814, USA.

Nucleic acids research

|July 2, 2024

概括

我们开发了IntePACE,一种新型的定向进化方法,以增强氨酸整合酶活性,以有效地传递大型转基因. 这一突破实现了高达80%的整合效率,加速了基因治疗的发展.

科学领域:

分子生物学分子生物学
生物技术是生物技术.
基因工程基因工程基因工程

背景情况:

目前用于生物医学干预的基因传递方法面临着低插入效率和依赖双链DNA断裂的挑战.
氨酸整合酶为在特定的基因组附着部位 (att) 插入大型DNA有效载荷提供了一个有希望的替代方案,在与基因组向技术 (如主要编辑) 结合时避免双链断裂.

研究的目的:

系统地提高PhiC31和Bxb1血清整合酶的活性,以有效地,有针对性地传递大型转基因.
开发一种新的定向进化策略,用于增强基因传递应用中的酶功能.

主要方法:

用菌体辅助连续进化 (PACE) 进行了广泛的突变发生,生成了一系列的血清酶整合酶变体库.
这种名为IntePACE的开发方法被用来快速进化PhiC31和Bxb1整合酶.
鉴定出协同突变,并将它们结合起来,产生过度活跃的整合酶变体.

主要成果:

产生了新型的高活性血清整合酶突变体,显著提高了DNA整合效率.
实现了多基因载荷的整合,包括编码威尔布兰德因子的15.7kB治疗性DNA,目标染色体的整合率高达80%.
通过使用IntePACE方法,可以快速和系统地改善整合活动.

结论:

工程化超活性整合酶和IntePACE定向进化策略代表了基因传递技术的重大进步.
这种方法有可能通过有效地插入大型治疗有效载荷来加速基因传递疗法的发展.
定向进化策略可以适应改善自然界中发现的其他新型整合酶.

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