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Cas9 RNP 物理化学分析用于增强CRISPR-AuNP组件和功能

Daniel D Lane¹, Karthikeya S V Gottimukkala^1,2, Rachel A Cunningham^1,2

¹Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.

bioRxiv : the preprint server for biology

|April 15, 2024

概括

研究人员开发了改进的CRISPR-gold纳米颗粒,用于在血液形成干细胞中的体内基因编辑. 这种方法增强了CRISPR-Cas9的传递和活性,有可能克服目前血液疾病的ex vivo疗法的局限性.

科学领域:

基因编辑和纳米医学用于血液学疾病.

背景情况:

目前针对β血症和状细胞贫血的CRISPR疗法需要对造血干细胞和原生细胞 (HSPC) 进行ex vivo操纵,涉及有毒电穿孔和化疗调节.
通过纳米载体进行CRISPR基因编辑工具的体内输送提供了一个有希望的替代方案,以减轻复杂性和毒性,但在克服HSPC限制因素方面面临挑战.
以前的CRISPR-金纳米粒子 (CRISPR-AuNP) 系统显示出不一致的Cas9活性,这是由于加载过程中RNA的不稳定性.

研究的目的:

开发一种携带CRISPR的黄金纳米粒子 (CRISPR-AuNP),以便在体内有效地将CRISPR-Cas核糖蛋白复合体 (RNP) 输送到HSPC中.
为了克服最初的CRISPR-AuNP配方中观察到的Cas9活性和负载不稳定的局限性.
为了提高纳米颗粒的稳定性和潜在的 in vivo 管理的特性.

主要方法:

在加载到金纳米粒子 (AuNP) 之前,Cas9和Cas12a RNP复合物的预制.
优化RNP加载化学和条件,以改善粒子结合.
用PEGylation修改纳米粒子外层,以提高稳定性和表面性能.

主要成果:

在不影响活动的情况下实现了39.6 ± 7.0 Cas9 RNP/AuNP的显著负载,并且Cas12a RNP/AuNP负载增加了10倍.
开发了第二代CRISPR-AuNP,其稳定性得到了改善,并且具有适合体内使用的水友性中性表面.
通过初级人类HSPCs证明了第二代CRISPR-AuNP的高体外吸收率 (72.5 ± 7.37%),尽管内体积累限制了基因编辑效率.

结论:

预制RNP和优化加载化学对于通过黄金纳米粒子在体内有效传递CRISPR-Cas9和Cas12a基因编辑系统至关重要.
开发的CRISPR-AuNP纳米配方显示了HSPC体内体内基因编辑的潜力,但需要进一步改进,以提高内体逃生和基因编辑效率.

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