PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells
PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells
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DOI：
10.1016/j.biomaterials.2020.120580
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发表时间：
2021-01-01
期刊：
影响因子：
14
通讯作者：
Eich, Christina
中科院分区：
文献类型：
--
作者：
Cruz, Luis J.;van Dijk, Thamar;Eich, Christina
CD34+造血干/祖细胞的体外基因编辑为开发多种恶性和非恶性疾病的新治疗方法提供了很好的机会。在HSPC中，通过电穿孔和/或病毒转导来传递CRISPR-复合体已经实现了高效的基因编辑，但细胞毒性是目前使用的方法的一个缺点。基于纳米颗粒(NP)的基因编辑策略可以进一步增强HSPC的基因编辑潜力，并为体内应用提供传递系统。在这里，我们开发了CRISPR/Cas9-PLGA-NPs，有效地包裹了Cas9蛋白、单个gRNA和一个荧光探针。Cas9和gRNA在最初的“爆发”释放之后是一个持续的释放模式。CRISPR/Cas9-PLGA-NPs被人HSPC摄取和加工，没有引起细胞毒性。CRISPR/Cas9-PLGA-NPs介导的γ-珠蛋白基因座的基因编辑使原代红系细胞中胎儿血红蛋白(HBF)表达增加。CRISPR/Cas9PLGA-NPs的研制为CRISPR组分的靶向HSPC提供了一种有吸引力的工具，并为体内治疗血红蛋白疾病和其他遗传性疾病提供了基础。
Ex vivo gene editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial 'burst' of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the gamma-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases.