Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability

Abstract

In vitro–transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Here, we investigated whether incorporation of naturally modified nucleotides into transcripts would confer enhanced biological properties to mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015–0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-α (IFN-α). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination.

Intratumoral Delivery of TriMix mRNA Results in T-cell Activation by Cross-Presenting Dendritic Cells

“…negatively affects its translation, implying that stronger TiDC activation could be obtained using "modified" mRNA that does not trigger PRRs (40)(41)(42). If PRRs are triggered, the effects observed in this study may be due to the delivery of mRNA rather than the delivery of TriMix mRNA.…”

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation

“…28 A modified nucleoside 59-triphosphate solution was used, in which pseudouridine-59-triphosphate (pseudo-UTP) and 5-methylcytidine-59-triphosphate (5-methyl-CTP) were substituted for UTP and CTP, respectively. 29 After transcription, uncapped RNA was purified using an RNeasy kit (Qiagen, Hilden, Germany) and a 59 cap structure and 39-poly(A) tail were added using the mScript kit. Transcripts were then repurified and quantified, and the success of the poly(A) tailing reaction was confirmed by agarose gel electrophoresis.…”

Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger

“…Two of the modified nucleosides, S2U and pseudouridine, seem to reduce detection by RIG-I and PKR as well [33,87]. Additionally, in 2008, pseudouridine was shown to increase mRNA translation capacity, by improving the overall stability of mRNA and avoiding PKR recognition [88,89]. In the same vein, Kormann et al indicated that replacement of 25% uridine and cytidine with s2U and m5C substantially reduced binding to PRRs and decreased innate immune activation, leading to an increased protein expression in vitro and in vivo [90].…”