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基于循环德克斯的量身定制的多聚素,以高效地传递基因组编辑分子

Toru Taharabaru¹, Takuya Kihara², Airi Obata²

¹Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan; Department of Biomedical Engineering, National University of Singapore, 15 Kent Ridge Crescent, Singapore 119276, Singapore.

Carbohydrate polymers

|November 8, 2023

概括

优化的多聚素 (PRXs) 有效地为CRISPR-Cas9基因组编辑提供Cas9核核蛋白. 结构性修改增强了细胞吸收,内体体逃逸和哺乳动物细胞的编辑效率.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
纳米技术纳米技术

背景情况:

对于有效的CRISPR-Cas9基因组编辑,Cas9核核蛋白 (RNP) 的直接细胞溶液输送至关重要.
基于循环德克斯的多素 (PRXs) 作为生物分子的转化载体具有潜力.
之前的工作开发了用于Cas9 RNP交付的氨基化PRX,显示了复杂化,内部化和核定位的前景.

研究的目的:

微调多步骤可转换PRX的结构,以增强Cas9 RNP交付.
为优化PRX介导的基因组编辑建立结构-属性关系.
为了研究连接物修饰对PRX载体性能的影响.

主要方法:

合成和表征了各种PRX衍生物,其分子长度和替代度被修改.
研究了控制PRX复杂化,细胞吸收和内体细胞逃逸的结构-属性关系.
在目标细胞中评估了携带Cas9 RNP的优化PRX的基因组编辑疗效.
嵌入的连接物修饰的β-环氧氨酸 (β-CD) 来增强细胞向和传递.

主要成果:

具有较长轴分子 (35 kDa PEG) 和低置换 (1.5 氨基基团/α-CD) 的PRX衍生物表现出优异的性能.
囊胺和二甲二胺的最佳比例 (≈1:1) 改善了细胞内动力学和编辑效率.
接体修饰的β-CD显著增强了PRX/Cas9 RNP复合体的细胞吸收和基因组编辑效应.
结构微调和连接体修饰共同导致了高效的基因组编辑.

结论:

多步骤可转换的PRX可以在结构上进行优化,以实现高效的Cas9 RNP交付.
特定的结构特征,包括分子长度和胺基修饰比率,对于增强内体逃脱和RNP释放至关重要.
灵原体修饰的β-CD集成进一步提高了基于PRX的CRISPR-Cas9基因组编辑系统的效率.

关键词:

这是一种Cas9核糖蛋白.基因组编辑基因组编辑细胞内输送细胞内输送多阶段可转换的聚甲.结构微调的结构微调.

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