Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.
Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.
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DOI：
10.1002/jev2.12343
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发表时间：
2023-09
影响因子：
16
通讯作者：
中科院分区：
文献类型：
--
作者：
簇状规则间隔回文重复序列(CRISPR)是一种具有巨大治疗潜力的基因编辑工具。最近，基于核糖核蛋白(RNP)复合体的CRISPR系统由于减少了非靶标编辑而获得了发展势头。这与细胞外小泡(EVS)作为一种治疗载体的出现不谋而合，因为它的免疫原性低，操作能力高。EVS是一种细胞来源的膜纳米颗粒，它介导了分子成分的细胞间转移。目前的技术通过EVS的生物发生将CRISPR RNP封装到EVS中，从而避免了直接对囊泡进行不必要的物理、化学或生物操作。在这里，我们确定了16种基于EVS的CRISPR RNP封装策略，每种策略都具有不同的遗传特征来封装CRISPR RNP。根据促进包囊过程的分子机制，基于基因融合将Cas9 RNP封装成病毒样颗粒的策略有6种，基于蛋白质拴系的策略有7种，基于sgRNA偶联包囊的策略有3种。此外，通过EVS生物发生将靶向部分掺入EVS膜表面，使其具有趋向性，并提高了对特定细胞类型的递送效率。靶向部分包括病毒包膜蛋白、含有配基多肽的重组蛋白、单链片段可变型抗体和整合素。然而，目前的策略仍然有一些限制，阻碍了它们在临床试验中的使用。其中，由于宿主免疫反应，引入病毒蛋白来包裹Cas9RNP引起了生物相容性的问题。未来的研究应集中于对不含病毒蛋白的EVS进行基因工程，提高EVS的递送特异性，并促进EVS基于同源基因的定向修复。然而，CRISPR RNP包裹和定向技术的集成将为基于EVS的CRISPR RNP在基因治疗和疾病治疗中的传递提供策略。
Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex‐based CRISPR systems have gained momentum due to their reduction of off‐target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell‐derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs‐based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus‐like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA‐coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single‐chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs‐based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs‐based delivery of CRISPR RNP in gene therapy and disease treatment.
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影响因子：
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