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.
Kevin J. Kauffman, J. Robert Dorkin, Jung H. Yang, Michael W. Heartlein, Frank DeRosa, Faryal F. Mir, Owen S. Fenton, and Daniel G. Anderson
Nano Lett 15(11): 7300-7306, 2015, doi: 10.1021/acs.nanolett.5b02497
Lipid nanoparticle (LNP) formulations have shown considerable efficacy in the intracellular delivery of small interfering RNA (siRNA). However, the potential of these LNPs for delivery of messenger RNA (mRNA) has only recently been realized. The nanoparticles are composed of four major lipids: (1) ionizable-amine-containing lipids or lipidoids, (2) a structural lipid such as a phospholipid, (3) cholesterol, and (4) and a polyethylene glycol conjugated lipid. Previous work has described the importance of the ratio of the four components in determining formulation efficacy for siRNA. In the present study, the ratio of the afore-mentioned components is optimized for the in vivo delivery of mRNA using Design of Experiment (DOE) methods. Employing Definitive Screening (DSD) and Fractional Factorial Design (FFD) methods, the number of individual experiments required to establish statistically significant trends in a large design space are reduced. In particular, in vivo screening of three quantitative parameters (low, intermediate, and high amounts) of each lipid in addition to varying the lipids tested (four phospholipid) would result in 324 individual experiments. However, by using DSD and FFD methods in a series of tests this number can be dramatically reduced to 38. By simultaneously varying the lipid composition and relative ratios, a 7-fold improvement in mRNA delivery efficacy was established. Improvements to the formulation include incorporation of fusogenic phospholipid 1,2-dioleoyl-sn -glycero-3-phosphoethanolamine (DOPE), and increasing the lipid:mRNA weight ratio. Interestingly, the optimized formulation of mRNA did not significantly change siRNA-mediated knockdown of FVII, suggesting the payload varies the particle design parameters.