Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
Cholesterol-rich lipid-mediated nanoparticles boost of transfection efficiency, utilized for gene editing by CRISPR-Cas9
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DOI：
10.2147/ijn.s199104
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发表时间：
2019-01-01
影响因子：
8
通讯作者：
Hosseinkhani, Saman
中科院分区：
文献类型：
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作者：
Hosseini, Elaheh Sadat;Nikkhah, Maryam;Hosseinkhani, Saman
目的：基因治疗已经成为一种很有前途的治疗方法，通过改变人的基因来治疗疾病。CRISPR-Cas9等相关工具的治疗潜力取决于靶向细胞的递送效率。许多转染试剂已经被设计出来，并且为此目的投入了大量的努力来开发载体。因此，本研究的目的是开发新型富含胆固醇的脂质纳米颗粒，以提高转染效率和血清稳定性。材料和方法：我们构建了两组分、三组分和四组分阳离子脂质体（CLs），以评估胆固醇结构域和二油基磷脂酰乙醇胺（dioleoyl phosphatidylethanolamine，一种促核脂质）以及胆固醇结构域内外PEG（聚乙二醇）片段位置对颗粒转染效率和其他性质的联合影响。在不同氮磷比下，通过凝胶阻滞试验评估脂质体形成和质粒DNA （pDNA）包裹（3,5,7）。研究脂质体的理化特性、细胞毒性、血清稳定性和内体逃逸能力，并用荧光素酶法测定转染电位。接下来，利用稳定表达GFP的HEK293细胞系，通过所选择的CL配方传递Cas9和sgRNA质粒，演示了对报告基因的编辑，转染效率最高。结果：在所设计的CLs中，四组份配方[DOTAP（1,2-二酰-3-三甲基氨丙烷）/DOPE/胆固醇/ choll - peg（胆固醇-聚乙二醇）]在N/ p3时转染率最高。最后，用该配方以N/P 3转染Cas9/sgRNA，敲除GFP报告基因的基因编辑效率为39%。结果还表明，该蛋白能完全保护质粒不受血清酶降解的影响，且无细胞毒性作用。结论：新型聚乙二醇化胆固醇结构域脂质体具有血清稳定性、较高的转染效率和内体释放性，可用于体内Cas9/sgRNA传递和其他未来基因治疗应用。
Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability.Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency.Results: Among the designed CLs, the four-component formula [ DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum.Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.