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.

Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose

“…RNA, particularly single-stranded RNA, is rapidly degraded by nucleases when injected in vivo (37). Although therapeutic RNA can be chemically modified to facilitate delivery and reduce immunogenicity (38)(39)(40)(41), no such modifications have been reported for replicons; this may reflect the sensitivity of the viral replicase to base modification (42)(43)(44) or to the secondary structure effects modifications may have on vital conserved RNA replication elements (45). We reasoned that nanoformulation could allow for the protection and functional delivery of replicon mRNA vaccines.…”

mRNA vaccines — a new era in vaccinology

“…Replacing rare codons with frequently used synonymous codons that have abundant cognate tRNA in the cytosol is a common practice to increase protein production from mRNA 29 , although the accuracy of this model has been questioned 30 . Enrichment of G:C content constitutes another form of sequence optimization that has been shown to increase steady-state mRNA levels in vitro 31 and protein expression in vivo 12 .…”

“…First, safety: as mRNA is a non-infectious, non-integrating platform, there is no potential risk of infection or insertional mutagenesis. Additionally, mRNA is degraded by normal cellular processes, and its in vivo half-life can be regulated through the use of various modifications and delivery methods 9–12 . The inherent immunogenicity of the mRNA can be down-modulated to further increase the safety profile 9,12,13 .…”

“…Additionally, mRNA is degraded by normal cellular processes, and its in vivo half-life can be regulated through the use of various modifications and delivery methods 9–12 . The inherent immunogenicity of the mRNA can be down-modulated to further increase the safety profile 9,12,13 . Second, efficacy: various modifications make mRNA more stable and highly translatable 9,12,13 .…”

“…The inherent immunogenicity of the mRNA can be down-modulated to further increase the safety profile 9,12,13 . Second, efficacy: various modifications make mRNA more stable and highly translatable 9,12,13 . Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm (reviewed in REFS 10,11).…”