[Cas蛋白向细胞外囊泡的随机包装]
[Stochastic Packaging of Cas Proteins into Exosomes].
作者信息
Ponomareva N I, Brezgin S A, Kostyusheva A P, Slatinskaya O V, Bayurova E O, Gordeychuk I V, Maksimov G V, Sokolova D V, Babaeva G, Khan I I, Pokrovsky V S, Lukashev A S, Chulanov V P, Kostyushev D S
机构信息
Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow, 119991 Russia.
Sirius University of Science and Technology, Sochi, 354340 Russia.
出版信息
Mol Biol (Mosk). 2024 Jan-Feb;58(1):160-170.
CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.
CRISPR/Cas系统是用于对遗传物质进行靶向操作的有前景的分子工具,如基因编辑、基因转录调控、表观基因组修饰等。虽然CRISPR/Cas系统在实验环境中已被证明对纠正遗传疾病、治疗传染病和癌症非常有效,但这些结果的临床转化受到缺乏高效的CRISPR/Cas递送载体的阻碍。基于有机和无机聚合物的现代合成纳米载体有许多缺点,包括毒性问题、缺乏靶向递送以及复杂且昂贵的生产流程。相比之下,外泌体是分泌的生物纳米颗粒,具有高生物相容性、物理化学稳定性以及穿越生物屏障的能力。早期临床试验发现外泌体注射没有毒性。近年来,外泌体被认为是CRISPR/Cas系统在体内的有前景的递送载体。本研究的目的是分析CRISPR/Cas随机包装到外泌体中对几种人类细胞系的效果。在这里,我们表明Cas9蛋白有效地定位于细胞内外泌体生物发生的区室中,但Cas9随机包装到外泌体中的比例非常低(约1%)。因此,Cas9蛋白的随机包装非常无效,不能用于基因编辑目的。需要开发将CRISPR/Cas系统加载到外泌体中的新型工具和技术。