School of Life Sciences, University of Science and Technology of China , Hefei, Anhui 230027, People's Republic of China.
Institutes for Life Sciences and School of Medicine, South China University of Technology , Guangzhou, Guangdong 510006, People's Republic of China.
ACS Nano. 2018 Feb 27;12(2):994-1005. doi: 10.1021/acsnano.7b07874. Epub 2018 Jan 9.
The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases. However, the inability to perform specific gene editing in targeted tissues and cells, which may cause off-target effects, is one of the critical bottlenecks for therapeutic application of CRISPR/Cas9. Herein, macrophage-specific promoter-driven Cas9 expression plasmids (pM458 and pM330) were constructed and encapsulated in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN). The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo. More importantly, after further encoding a guide RNA targeting Ntn1 (sgNtn1) into the plasmid, the resultant CLAN successfully disrupted the Ntn1 gene in macrophages and their precursor monocytes in vivo, which reduced expression of netrin-1 (encoded by Ntn1) and subsequently improved type 2 diabetes (T2D) symptoms. Meanwhile, the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by the CD68 promoter. This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/Cas9 system.
CRISPR/Cas9 基因编辑技术有望用于多种疾病的治疗。然而，无法在靶向组织和细胞中进行特定的基因编辑，这可能导致脱靶效应，是 CRISPR/Cas9 治疗应用的关键瓶颈之一。在此，构建了巨噬细胞特异性启动子驱动的 Cas9 表达质粒（pM458 和 pM330），并将其包裹在阳离子脂质辅助的 PEG-b-PLGA 纳米粒（CLAN）中。所得的 CRISPR/Cas9 质粒包封的纳米粒能够在体外和体内特异性地在巨噬细胞及其前体细胞单核细胞中表达 Cas9。更重要的是，进一步将靶向 Ntn1 的向导 RNA（sgNtn1）编码到质粒中后，所得的 CLAN 成功地在体内破坏了巨噬细胞及其前体细胞单核细胞中的 Ntn1 基因，降低了 netrin-1（由 Ntn1 编码）的表达，随后改善了 2 型糖尿病（T2D）症状。同时，由于 CD68 启动子特异性表达 Cas9，Ntn1 基因未在其他细胞中被破坏。该策略为 CRISPR/Cas9 系统的体内特异性基因编辑提供了替代途径。
