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.