Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin

Abstract

Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10-100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.

Figure 1

Codon-optimized, pseudouridine-containing murine erythropoietin (EPO) mRNA yields the highest level of translation. Human dendritic cells (DCs) were treated with TransIT-complexed in vitro-transcribed mRNA (0.1 µg/well) with the wild-type or codon-optimized sequence of murine EPO containing uridine (U-mRNA) or pseudouridine (Ψ-mRNA). The levels of EPO in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA) 24 hours later. Error bars are standard errors of the mean (SEM). The data shown is one of three representative experiments.

Figure 2

Physiologic responses of mice injected with erythropoietin (EPO) mRNA. (a) Administration of EPO mRNA increases serum EPO levels. Adult mice were injected intraperitoneally (i.p.) with TransIT-complexed mRNA (0.1 µg) coding for murine EPO containing pseudouridine (EPO Ψ-mRNA) or containing uridine (EPO U-mRNA) or coding for firefly luciferase and containing pseudouridine (luc Ψ-mRNA) or with 3 µg of recombinant murine EPO (rmEPO) protein. Serum EPO levels were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points. Five animals per condition were analyzed. (b) Increased reticulocyte count following a single injection of EPO mRNA. Mice received a single i.p. injection of the indicated amounts of EPO Ψ-mRNA, luc Ψ-mRNA, or EPO U-mRNA complexed with TransIT or human EPO protein (n = 3–11). Reticulocytes were counted at the indicated days using a 5 µl blood sample. Mice were injected with TransIT-complexed mRNA once with 1.0 µg RNA (c), or weekly (indicated by arrows) with 0.1 µg mRNA (d). Hematocrits were measured using 20 µl of blood. Five animals per group were analyzed. (e) Mice were injected with 1.0 µg of murine EPO mRNA that contained either pseudouridine or uridine, or were left uninjected. Murine IFN-α levels were measured in the plasma at 6 hours post-injection by ELISA. Individual data points and the group average (red line) are shown. Error bars are SEM.

Figure 3

Pseudouridine-containing rhesus erythropoietin (EPO) mRNA increases circulating levels of EPO in macaques. Rhesus macaques (n = 4) were injected intraperitoneally (i.p.) with TransIT-complexed rhesus EPO Ψ-mRNA (0.012 mg/kg). Serum EPO levels were measured by enzyme-linked immunosorbent assay (ELISA). Error bars are SEM.