Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

中文翻译：

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用化合价控制的DNA纳米结构工程化的细胞外囊泡可提供用于基因治疗的CRISPR / Cas9系统。

细胞外小泡（EVs）有望在整个体内转运CRISPR–Cas9 RNA引导的内切核酸酶（RNP）。然而，电动汽车的细胞选择性递送仍然是一个挑战。在这里，我们设计了与DNA适体偶联的化合价控制的四面体DNA纳米结构（TDN），并通过胆固醇锚定将化合价控制的TDNs装载在EV表面，以实现特定的细胞靶向。研究了不同比例的适体/胆固醇从1：3至3：1在TDN中对装饰电动车的靶向功效。具有一个适体和三个胆固醇锚定（TDN1）的TDN可以有效地促进EV在培养的HepG2细胞和人类原发性肝癌衍生的类器官以及异种移植肿瘤模型中的肿瘤特异性蓄积。TDN1-EVs在细胞内递送RNP成功地实现了其随后的基因组编辑，从而导致特定细胞中GFP或WNT10B的下调。该系统最终用于降低WNT10B的蛋白质表达，从而显着抑制了肿瘤的生长在体外，离体和体内，并且可以扩展到其他治疗靶标。本研究为在表面标记上适体的定向展示和基于EVs的Cas9传递提供了平台，这为将来的细胞选择性基因编辑提供了有意义的想法。