Yin Hao, Song Chun-Qing, Suresh Sneha, Wu Qiongqiong, Walsh Stephen, Rhym Luke Hyunsik, Mintzer Esther, Bolukbasi Mehmet Fatih, Zhu Lihua Julie, Kauffman Kevin, Mou Haiwei, Oberholzer Alicia, Ding Junmei, Kwan Suet-Yan, Bogorad Roman L, Zatsepin Timofei, Koteliansky Victor, Wolfe Scot A, Xue Wen, Langer Robert, Anderson Daniel G
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Nat Biotechnol. 2017 Dec;35(12):1179-1187. doi: 10.1038/nbt.4005. Epub 2017 Nov 13.
Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9-sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.
在体内使用Cas9-sgRNA进行高效基因组编辑需要借助病毒递送系统，而这在临床应用中存在局限性。开发其他RNA疗法的转化研究表明，对RNA进行明智的化学修饰可通过降低核酸酶降解敏感性来提高治疗效果。在Cas9-sgRNA复合物结构的指导下，我们确定了sgRNA中可在保持或增强基因组编辑活性的同时进行修饰的区域，并开发了一套适用于体内应用的最佳化学修饰方法。使用这些增强型sgRNA（e-sgRNA）和编码Cas9的mRNA的脂质纳米颗粒制剂，我们发现对小鼠进行单次静脉注射可诱导肝脏中超过80%的Pcsk9编辑。血清Pcsk9水平降至检测不到，动物体内胆固醇水平显著降低约35%至40%。该策略可能使临床环境下在肝脏中实现基于Cas9的非病毒基因组编辑成为可能。
