Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

Abstract

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies.

Figure 1

Sequence-engineered mRNA yields high protein expression in vitro. (a) Engineered, unmodified luciferase mRNA outperforms its nucleoside-modified counterparts. HeLa cells were lipofected in triplicate with the indicated amounts of a luciferase-encoding mRNA that was either unmodified or harbored the given nucleoside modifications. Luciferase expression was quantified 24 hours after transfection. (b) Engineered but unmodified mouse Epo mRNA is superior to nucleoside-modified variants. HeLa cells were lipofected in triplicate with different amounts of an erythropoietin-encoding mRNA that was either unmodified or modified by incorporation of the indicated nucleosides. Erythropoietin levels in the supernatant were quantified 24 hours after transfection. (c) Pseudouridine incorporation improves the expression of an unmodified basic luciferase mRNA. HeLa cells were transfected in triplicate with different amounts of a basic luciferase-encoding mRNA either being unmodified or harboring pseudouridine instead of the unmodified nucleotide. Protein expression was quantified 24 hours after transfection. unmod, engineered mRNA harboring the nucleotides A, U, G, and C; ψ, engineered mRNA in which pseudouridine replaces U; ψ+5mC, engineered mRNA in which U and C are replaced by pseudouridine and 5-methly-cytosine, respectively; basic, mRNA comprising a cap, a codon-optimized open reading frame, an α-globin 3'-UTR as well as a polyA.

Figure 2

Sequence engineering of mRNA enables high level protein expression in vivo. (a) Engineered, unmodified mouse Epo mRNA yields higher protein levels in mice than the corresponding pseudouridine modified sequence. 1 μg of either unmodified or pseudouridine modified mRNA encoding erythropoietin was complexed with TransIT and intraperitoneally injected into mice. Serum erythropoietin (EPO) levels were determined at different times (1, 2, or 3 days) after administration. (b) Low nanogram doses of engineered, unmodified mouse Epo mRNA give rise to substantial protein levels in murine serum. The indicated amounts of TransIT-complexed unmodified Epo mRNA were administered intraperitoneally. As a control, 100 U of recombinant human EPO protein (hEPO) were given i.p. Serum EPO levels were determined 24 hours after treatment. unmod, engineered mRNA harboring the nucleotides A, U, G, and C; ψ, engineered mRNA in which pseudouridine replaces U. n = 4 for all groups.

Figure 3

Sequence engineered Epo mRNA elicits physiological effects in mice. (a) Sequence-engineered mouse Epo mRNA elicits strong reticulocyte responses in mice. Mice were intraperitoneally injected with either TransIT-complexed mRNA (1 μg) or recombinant erythropoietin (EPO) protein (hEPO: 100 U, mEPO: 800 ng). mRNA was either unmodified or harbored pseudouridine. The level of reticulocytes was determined 4 days after treatment. (b) Low nanogram doses of engineered but unmodified mouse Epo mRNA elicit substantial reticulocyte responses in mice. The indicated doses of TransIT-complexed Epo mRNA or 100 U of hEPO were intraperitoneally administered to mice. Reticulocytes were quantified 4 days after injection. (c) Sequence-engineered mouse Epo mRNA substantially increases the hematocrit in mice. Mice were treated with either TransIT-complexed mRNA (1 μg) or recombinant EPO protein (hEPO: 100 U, mEPO: 800 ng) on day 0 and 14. The hematocrit was measured on day 18. mRNA was either unmodified or harbored pseudouridine. unmod, engineered mRNA harboring the nucleotides A, U, G, and C; ψ, engineered mRNA in which pseudouridine replaces U; hEPO, recombinant human Epo protein; mEPO, recombinant murine Epo protein. n = 4 for all groups.

Figure 4

Repeated treatments with engineered, unmodified Epo mRNA do not affect the immune status of mice inappropriately. (a) A single intraperitoneal injection of engineered, unmodified mouse Epo mRNA does not cause any substantial cytokine secretion. Mice were treated with different amounts of TransIT-complexed mouse Epo mRNA, vehicle only, or injection buffer. Six hours after injection, blood was collected and analyzed for the levels of various cytokines. As a positive control (pos. ctrl), mice were injected with an immunostimulatory RNA solution intramuscularly. (b) Repeated intraperitoneal injections of engineered but unmodified mouse Epo mRNA does not lead to any substantial cytokine secretion. Mice were treated twice a week for 3 weeks with different amounts of TransIT-complexed mRNA, vehicle only, or an injection buffer. Six hours after the last injection, blood was collected and analyzed for the levels of various cytokines. As a positive control, animals received a single dose of an immunostimulatory RNA solution intramuscularly. (c) Repeated intraperitoneal injections of engineered but unmodified mouse Epo mRNA did not induce an erythropoietin (EPO)-specific antibody response. Mice were treated twice a week for 3 weeks with different amounts of TransIT-complexed mRNA or vehicle only. Four weeks after the last administration, blood was collected and analyzed for the presence of EPO-specific antibodies. For comparison, an anti-EPO-antibody from rat was used as positive control. n = 5 for pos. ctrl for cytokine measurements, n = 4 for all other groups.

Figure 5

Engineered, unmodified Epo mRNA allows long-term/continued treatment of mice. (a) Protein yield from intraperitoneally administered mouse Epo mRNA is not affected by repeated treatments. Mice were repeatedly injected with 1 μg of either unmodified or pseudouridine-modified mRNA as well as 0.1 μg of unmodified mRNA at an interval of 2 weeks. Plasma erythropoietin (EPO) levels were determined 24 hours after each treatment. (b) Intraperitoneally injected mouse Epo mRNA increases reticulocyte counts even after multiple dosing. Mice received multiple doses of TransIT-complexed mRNA (1 μg, either unmodified or pseudouridine-modified) or mEPO (0.8 μg) at a biweekly interval starting on day 0 and were analyzed for reticulocytes 4 days after treatments. (c) Repeated intraperitoneal injections of Epo mRNA elicit a strong and sustained increase of the hematocrit. Mice received multiple doses of TransIT-complexed mRNA (1 μg, either unmodified or pseudouridine-modified) or mEPO (0.8 μg) at a biweekly interval starting on day 0 and were analyzed for the hematocrit at various times. n = 4 for all groups.

Figure 6

Engineered, unmodified Epo mRNA can elicit systemic physiological responses in swine and nonhuman primates. (a) Intravenous mRNA injection into pigs gives rise to high serum erythropoietin (EPO) levels as well as to substantial physiological effects. Animals received 1.3 mg of lipid nanoparticle (LNP) encapsulated porcine Epo mRNA (0.065 mg/kg) on day 0 and were analyzed for EPO levels and hematological parameters at various times. Protein levels before treatment were below the limit of detection of the assay. (b) Intravenous mRNA injection into macaques increases EPO levels, reticulocyte numbers as well as the hematocrit. Animals received 100 μg of LNP encapsulated M. fascicularis Epo mRNA (0.037 mg/kg) and were analyzed for EPO levels and hematological parameters before and at various times after treatment. Distinct symbols are assigned to individual animals. Pre, prevalue before treatment, n = 3 for pig experiment, n = 4 for macaque study.