Hao Yin, Chun-Qing Song, Joseph R Dorkin, Lihua J Zhu, Yingxiang Li, Qiongqiong Wu, Angela Park, Junghoon Yang, Sneha Suresh, Aizhan Bizhanova, Ankit Gupta, Mehmet F Bolukbasi, Stephen Walsh, Roman L Bogorad, Guangping Gao, Zhiping Weng, Yizhou Dong, Victor Koteliansky, Scot A Wolfe, Robert Langer, Wen Xue, Daniel G Anderson
Nature Biotechnology 34: 328-333, doi:10.1038/nbt.3471
CRISPR-Cas9-mediated gene editing allows for precise correction of genetic diseases when appropriately administered. Cas9 complexed with RNA-guide strands (sgRNA) recognizes target sequences and creates double-stranded DNA breaks. Endogenous repair mechanisms including non-homology end joining, and precise homology-directed repair are then activated to correct these breaks. For gene correction purposes, the Cas9 protein, in addition to the sgRNA and exogenous DNA template, need to be delivered to the nucleus. However, Cas9 persistence in the nucleus has the potential to cause non-specific DNA damage. Delivery of Cas9 mRNA ensures robust, yet short-term expression of the functional product, while co-delivery of the sgRNA and the DNA template using an adeno-associated viral (AAV) vector ensures nuclear localization. In this study, the various components of CRISPR are delivered to target cells using lipid nanoparticles (LNPs) for Cas9 mRNA, and an AAV vector for the sgRNA, and DNA template. In particular, in vivo characterization of the LNP-AAV combination was targeted against the mouse model of hereditary tyrosinemia, which is caused by a G-A point mutation in the gene encoding fumarylacetoacetate hydrolase (FAH). The combination treatment showed a gene correction efficiency of > 6% hepatocytes after a single dose showing the potential clinical utility of genome editing technology.