Mice were IV-injected with LNPs carrying either unmodified or modified luciferase mRNA at a dose of 0.75 mg/kg. Six hours after injection, organs were harvested and imaged for bioluminescence by IVIS. (A-C) Protein expression resulting from C12-200 delivery was enhanced by the ψ, m1ψ, and m5C/ψ modifications, with the vast majority of the improvement in the spleen. (D-F) mRNA modifications did not improve overall expression for cKK-E12 mRNA delivery with statistical significance. Nonetheless, spleen expression improved with m1ψ and m5C/ψ modifications. (G-I) 200Oi10-mediated protein expression benefitted the most from nucleoside modifications, particularly with the m1ψ modification. Improvements in spleen expression were dramatic. (J-L) When formulated into ZA3-Ep10 LNPs, ψ and m1ψ modifications enhanced expression in the spleen, with very little improvement in the lungs. Error bars represent s.d. (n = 3; *p< 0.05, **p< 0.01, ***p<0.001; unpaired t-test relative to unmodified).