Fig. 4: In Vivo Expression of FLuc mRNA in LNPS with ionizable lipids DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA, and DODAP.

FLuc mRNA LNPs (5 µg) with NP lipid:mRNA ratio 4 were injected intramuscular (IM) or intravenous (IV) administration and imaged at 4 h and 24 h post-injection. a FLuc expression 4 h after IM administration and b 4 h after IV administration showing ROIs used to calculate Total Flux (photons/second). Scale bar is Radiance (p/sec/cm²/sr). FLuc expression (Total Flux) for c IM administration (†p < 0.055) and d IV administration at 4 h and 24 h and e the ratio of Liver to Muscle photon flux for IM administration. KC2 was the most potent LNP for IM administration while MC3 with a pKa 0.4 points lower than KC2 was the most potent for IV administration. The ratio of liver to muscle expression after IM administration, an undesirable off-target effect, is highest for MC3 and is associated with a lower pKa, lower pI and a more negatively charged LNP than KC2. f In vitro FLuc expression was strongly predictive (R² = 0.998, p < 0.0001) of In Vivo FLuc IM expression at all doses shown here for 25 ng dose only, but not predictive of In Vivo FLuc IV expression (R² = 0.446, p = 0.22). Off-target expression in liver was also correlated to potency. Data and mean are shown in graphs. For IM administration of DLin, DODMA, and DODAP, expression in liver was too low for quantification.