Sousa Diana A, Gaspar Ricardo, Ferreira Celso J O, Baltazar Fátima, Rodrigues Ligia R, Silva Bruno F B
CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
INL-International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, 4715-330 Braga, Portugal.
Pharmaceutics. 2022 May 19;14(5):1087. doi: 10.3390/pharmaceutics14051087.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome−DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.
成簇规律间隔短回文重复序列（CRISPR）和CRISPR相关核酸酶9（Cas9）基因编辑为癌症、心血管疾病、神经疾病和免疫疾病等具有遗传病因的疾病治疗提供了令人兴奋的新治疗可能性。然而，其临床转化受到缺乏安全、通用和有效的非病毒递送系统的阻碍。在此，我们报告了两种用于CRISPR/Cas9基因递送的阳离子脂质体-DNA系统（即脂质体复合物）的制备和应用。为此，使用了两种类型的阳离子脂质（DOTAP，单价，和MVL5，多价，标称电荷为+5e），以及三种类型的辅助脂质（DOPC、DOPE和单油酸甘油酯（GMO））。我们证明，编码Cas9和单向导RNA（sgRNA）的质粒，由于其尺寸较大（>9 kb），通常难以转染，但可以通过基于MVL5的脂质体复合物成功转染到HEK 293T细胞中。相比之下，基于DOTAP的脂质体复合物导致非常低的转染率。基于MVL5的脂质体复合物具有从溶酶体逃逸的能力，这可能解释了其优异的转染效率。关于基因编辑，基于MVL5的脂质体复合物实现了有前景的绿色荧光蛋白敲除水平，对于电荷比（+/-）为10时，敲除率超过35%。尽管敲除效率与Lipofectamine 3000®商业试剂相当，但基于MVL5的制剂中非特异性基因敲除更为明显，这可能是由于这些制剂具有相当大的细胞毒性。总之，这些结果表明，基于多价脂质的脂质体复合物是有前景的CRISPR/Cas9质粒递送载体，通过进一步优化和功能化，可能成为合适的体内递送系统。
