Dr. Katalin Karikó and Dr. Drew Weissman were jointly awarded the 2023 Nobel Prize in Physiology or Medicine “for their discoveries concerning nucleoside base modifications that enabled the development of effective mRNA vaccines against COVID-19.” This announcement went on to say that, in studying why synthetic mRNA is inflammatory, “they produced different variants of mRNA, each with unique chemical alterations in their bases, which they delivered to dendritic cells. The results were striking: The inflammatory response was almost abolished when base modifications were included in the mRNA.”
These seminal results were published in 2005, fifteen years before the COVID-19 pandemic. In further studies published in 2008 and 2010, Karikó and Weissman showed that the delivery of mRNA generated with base modifications markedly increased protein production compared to unmodified mRNA. “Through their discoveries that base modifications both reduced inflammatory responses and increased protein production, Karikó and Weissman had eliminated critical obstacles on the way to clinical applications of mRNA.”
The transformative nature of these discoveries is apparent from the following discussion, first about mRNA, and then alternative types of RNAs as therapeutics, all made possible by the incorporation of nucleoside modifications. These therapeutics broadly cover protective vaccines, protein-based therapies, and gene editing.
The three publications by Karikó and Weissman mentioned above have collectively received ~4,000 citations in other publications, which indicates a very high level of impact. A large majority of these citing publications deal with research and development of nucleoside-modified mRNAs as a new class of vaccines for protection against infectious diseases, as comprehensively reviewed elsewhere. Another comprehensive review of nucleoside-modified mRNA focuses on cancer vaccines. Remarkably, there are currently 450 clinical studies indexed to mRNA vaccines listed in the NIH Clinical Trials searchable database. A recent review of nucleoside-modified mRNA for protein-replacement therapy is also available.
For vaccines, one or more disease-associated antigens (or neoantigens for cancer) must first be identified and then optimally encoded in mRNA, which is commonly delivered in lipid nanoparticles (LNPs) or other polymer complexes. A 2021 review of LNPs for delivery of mRNA has already been accessed ~490,000-times as of this writing.
Fittingly, Weissman and collaborators recently reported an example of a muti-antigen (multivalent) vaccine for protection against malaria, which in 2021 led to 619,000 deaths, mostly young children. This novel bivalent vaccine is comprised of one LNP containing two N1-methylpseudouridine (m1ψ)-modified mRNAs, each encoding an antigen from Plasmodium falciparum, the mosquito-borne organism that causes malaria. This combined targeting of both infection-antigen and transmission-antigen is critical for achieving the elimination of malaria. Weissman and other collaborators have expanded the multivalent approach to one LNP containing 20 (a new record!) m1ψ-mRNA-encoded antigens representing all of the known strains of influenza virus, thus enabling the possibility of the first universal flu vaccine.
As for recent published work by Karikó, she and others at BioNTech, in collaboration with Genevant reported mRNA with ψ or m1ψ modifications for LNP delivery of an encoded enzyme to treat argininosuccinic aciduria. This inherited metabolic disorder occurs in 220,000 births worldwide, leading to severe medical problems. This and other protein-replacement therapies have importantly benefitted from nucleoside-modified mRNAs because of their negligible or lower inflammatory side effects, thus making it possible for patients to tolerate repeated doses of the mRNA-encoded missing or defective protein.
In closing this section, it should be mentioned that TriLink’s long-standing expertise with, and supply of, modified nucleotide triphosphates (NTPs) used for in vitro transcription (IVT) of mRNA fueled the early work by Karikó, Weissman, and then others. TriLink’s involvement also led to identification of 5-methoxyuridine (5-moU)-modified mRNA for muting inflammatory response and increasing translation. In addition, TriLink’s discovery of co-transcriptional synthesis of mRNA using nucleoside-modified trimers for increased 5’-capping efficiency led to its line of now widely employed CleanCap® mRNA capping reagents. Continuation of this R&D recently led to the launch of the new CleanCap® M6 reagent, which increases protein translation by ˃30% in a comparative study.
The transformative effect of nucleoside modifications in RNA therapeutics has been recently extended to self-amplifying RNA (saRNA), which differs from traditional mRNA by encoding viral components for amplification of its RNA resulting in more copies of the message to be translated into the co-encoded protein of interest. Because saRNA can therefore produce more protein of interest compared to mRNA, a dose of saRNA can exhibit higher potency relative to the same dose of traditional, non-amplified mRNA.
Despite this dosing advantage, saRNA has been heretofore studied without nucleoside modifications, due to concerns for inhibition of amplification by such modifications. Remarkably, this concern has been countered by the following new experimental observations.
McGee et al. (2023) used CleanCap® AU Reagent and nucleoside-modified NTPs from TriLink to co-transcriptionally synthesize 26 different mCherry reporter-saRNAs wherein a modified nucleoside completely replaced its unmodified counterpart. This panel of 26 modified nucleosides were comprised of 6 A, 6 C, 4 G, 5 U and 5 ψ analogs. LNP-mediated transfection into a T-cell line led to identification of 3 modifications that afforded significantly elevated levels of mCherry-positive cells, compared to the unmodified control, namely, 5-hydroxymethylcytidine, 14-fold; m5C, 10-fold; and 5-methyluridine (m5U), 8-fold.
This better performance with m5C was also found in vivo. Injection of mice with LNP-formulated m5C-saRNA encoding firefly luciferase showed ~4-fold higher bioluminescence compared to m1ψ-modified mRNA after 7 days and persisted at this increased level up to 28 days. In addition, gene expression analysis revealed that, whereas the unmodified saRNA induces a significant increase in the expression of immunogenic interferon genes after 6 hours in peripheral blood mononuclear cells from human donors, incorporation of 5-hydroxymethylcytidine or m5C led to a large reduction in the expression of these early IFN genes.
These potency and anti-inflammatory improvements found for 100% m5C-modified saRNA were independently confirmed by Aboshi et al. (2023) in a Phase 1 clinical study of a saRNA encoding the SARS-CoV-2 spike protein. Interestingly, this was achieved with only 5% of C replaced with m5C, using a 5:95 molar ratio of m5C:C triphosphates from TriLink.
RNA therapeutics has been further transformed by nucleoside modifications in the context of circular RNA (circRNA). By way of introduction, and in contrast to traditional linear mRNA, naturally occurring circRNAs do not have a 5’-cap for initiation of ribosome-mediated translation and do not have a 3’ poly(A) tail for stabilization. Consequently, circRNAs do not code for proteins and instead have other biological functions, as discussed here.
To engineer circRNAs vectors for encoded protein delivery, Chen et al. (2023) designed a high-throughput, protein-reporter (NanoLuc) assay system to screen large numbers of circRNA designs readily assembled from modular “parts.” All the parts were systematically varied at the sequence level to obtain many variants for empirical screening in the assay system, which identified optimized saRNA parts for producing encoded protein.
Because it was important to suppress undesired recognition of the optimized circRNAs by the immune system, all candidate circRNAs screened incorporated 5% N6-methyladenosine (m6A) during IVT synthesis using a 5:95 molar ratio of m6A:A triphosphates from TriLink. The resultant best performing circRNA design was then applied to the encoding of human erythropoietin (hEPO), which stimulates the production of red blood cell production.
It was found that hEPO-induced counts of new red blood cells were significantly increased in mice 1 week after a single dose of 5% m6A hEPO-circRNA, compared to the same dose of CleanCap® 100% m1ψ-modified hEPO mRNA. This and other data showed that optimally engineered circRNAs can express encoded proteins at levels comparable to modified mRNAs in vivo but with greater duration.
TriLink congratulates Dr. Karikó and Dr. Weissman for their pioneering discoveries in nucleoside-modified mRNA and look forward to the promising role of mRNA and RNA in therapeutics, vaccines, and personalized medicine.