College of Pharmacy, Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon, 16499, South Korea.
Moogene Medi Co. Ltd., Korea Bio Park, Daewangpangyo-ro 700, Seongnam, 13488, South Korea.
J Nanobiotechnology. 2019 Jan 29;17(1):19. doi: 10.1186/s12951-019-0452-8.
Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly.
In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses.
Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.
由于蛋白质 Cas9 的不稳定性和低效率，其体内基因编辑治疗法具有实际的局限性。为了克服这些障碍并提高稳定性，我们设计了一种主要由卵磷脂组成的纳米载体，该载体可以通过聚合物融合自组装，有效地靶向肝脏疾病，并将 Cas9 与单链向导 RNA（sgRNA）核糖核蛋白（Cas9-RNP）复合物封装起来。
在这项研究中，我们优化了一种针对二肽基肽酶-4 基因（DPP-4）的 sgRNA 序列，以调节胰高血糖素样肽 1 的功能。然后，我们将我们的纳米载体 Cas9-RNP 复合物直接注射到 2 型糖尿病（T2DM）db/db 小鼠体内，该复合物可显著破坏 T2DM 小鼠中 DPP-4 基因的表达。DPP-4 酶活性的下降伴随着血糖水平的正常化、胰岛素反应的增强以及肝肾功能损伤的减轻。这些结果与西他列汀（一种目前需要重复剂量的化学 DPP-4 抑制治疗药物）相似。
我们的结果表明，具有治疗性 Cas9-RNP 的纳米脂质体载体系统具有很大的潜力，可作为改善人类肝脏疾病的基因组编辑治疗的平台。
