Figure 4. Ψ-modified mRNAs are nonimmunogenic and have a higher translational capacity than unmodified mRNA in mice.

In vitro-transcribed capTEVlucA₅₀ (1,866 nt) with or without Ψ modifications were extended with long 3′-end poly(A) tail (+A_n) using poly(A) polymerase. Aliquots (1 μg) of mRNAs before and after poly(A) tailing were analyzed on denaturing agarose gel followed by ethidium bromide staining and ultraviolet (UV) illumination. The estimated lengths of poly(A) tails are ~200 nt. mRNAs used for the animal studies are indicated by asterisks. (b) Sixty-microliter aliquots of lipofectin-complexed mRNA (0.3 μg capTEVluc-A_n/mouse) containing Ψ modifications were administered by caudal vein injection. Animals were killed at 2 and 4 hours postinjection and luciferase activities were measured in aliquots (1/10th) of organs homogenized in lysis buffer. Values represent luciferase activities in the whole organs. Results with capRen were quantitatively identical except that liver and kidney had high endogenous Renilla luciferase-like activities (data not shown). (c,d) Lipofectin-complexed capTEVluc-A_n (0.3 μg/60 μl/animal) with or without Ψ modifications were intravenously (IV) delivered to mice. Animals were killed at 1, 4, and 24 hours postinjection and one-half of their spleens were processed for (c) luciferase measurements (d) while the other half for RNA analyses. Luciferase activities were measured in aliquots (1/5th) of the homogenate made from half of the spleens. Plotted values represent luciferase activities in the whole spleen and are expressed as the mean ± SEM. (n = 3 or 4/point). A similar pattern of expression was obtained in time-course experiments using capRen (data not shown). Dotted line represents background activity measured in spleen samples from animals injected only with lipofectin. (d) Aliquots of RNA (2 μg) isolated from the other half of spleens were analyzed by northern blot for luciferase, tumor necrosis factor-α (TNF-α) and β-actin. Autoradiograms and the corresponding ethidium bromide-stained, UV-visualized 28S and 18S rRNAs are presented. For luciferase, radiograms obtained after short and long exposure times (3 hours and 2 days) are shown. Animals, uninjected (control) and IV injected with uncomplexed lipofectin (lipofectin), were also processed. RNAs containing uridines (U) or pseudouridines (Ψ) are indicated. (e) The indicated amounts of lipofectin-complexed nucleic acids, capTEVluc-A_n mRNA with or without Ψ constituents and pCMVluc plasmid DNA in a volume of 60 μl/animal were delivered by IV injection into mice. Animals injected with mRNA or plasmid DNA were killed at 6 or 24 hours postinjection, respectively, and luciferase activities were measured in aliquots (1/10th) of their spleens homogenized in lysis buffer. Presented values were adjusted to signify luciferase activities in the whole organs (n = 3–5/point). The value from each animal is shown and the short horizontal lines indicate the mean; ND, not detectable. (f) Serum samples, collected during killing (6 hours postinjection) from the same animals that were processed for luciferase assessment shown in e, were analyzed by enzyme-linked immunosorbent assay which revealed that 3 μg of unmodified mRNA induced a higher level of interferon-α (IFN-α) than 3 μg of Ψ-modified mRNA did (P < 0.001). The levels of IFN-α induced by 3 μg of Ψ-modified mRNA were similar to those obtained when animals were injected with uncomplexed lipofectin (P = 0.19). Values are expressed as the mean ± SEM (n = 3 or 5 animals/group).