Nucleoside-modified Messenger RNA
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A nucleoside-modified messenger RNA (modRNA) is a synthetic messenger RNA (mRNA) in which some nucleosides are replaced by other naturally modified nucleosides or by synthetic nucleoside analogues. modRNA is used to induce the production of a desired protein in certain cells. An important application is the development of mRNA vaccines, of which the first authorized were COVID-19 vaccines (such as Comirnaty and Spikevax).


Background

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
is produced by synthesising a
ribonucleic acid Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydr ...
(RNA) strand from
nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecul ...
building blocks according to a deoxyribonucleic acid (DNA) template, a process that is called transcription. When the building blocks provided to the
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...
include non-standard nucleosides such as pseudouridine — instead of the standard adenosine, cytidine, guanosine, and uridine nucleosides — the resulting mRNA is described as nucleoside-modified. Production of protein begins with assembly of ribosomes on the mRNA, the latter then serving as a blueprint for the synthesis of proteins by specifying their amino acid sequence based on the
genetic code The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
in the process of
protein biosynthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside cells, balancing the loss of cellular proteins (via degradation or export) through the production of new proteins. Proteins perform a number of critical ...
called
translation Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
.


Overview

To induce cells to make proteins that they do not normally produce, it is possible to introduce heterologous mRNA into the
cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...
of the cell, bypassing the need for transcription. In other words, a blueprint for foreign proteins is "smuggled" into the cells. To achieve this goal, however, one must bypass cellular systems that prevent the penetration and translation of foreign mRNA. There are nearly-ubiquitous
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
s called ribonucleases (also called RNAses) that break down unprotected mRNA. There are also intracellular barriers against foreign mRNA, such as innate immune system receptors, toll-like receptor (TLR) 7 and TLR8, located in endosomal membranes. RNA sensors like TLR7 and TLR8 can dramatically reduce protein synthesis in the cell, trigger release of
cytokine Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in a ...
s such as interferon and TNF-alpha, and when sufficiently intense lead to
programmed cell death Programmed cell death (PCD; sometimes referred to as cellular suicide) is the death of a cell (biology), cell as a result of events inside of a cell, such as apoptosis or autophagy. PCD is carried out in a biological process, which usually confers ...
. The inflammatory nature of exogenous RNA can be masked by modifying the nucleosides in mRNA. For example, uridine can be replaced with a similar nucleoside such as pseudouridine (Ψ) or N1-methyl-pseudouridine (m1Ψ), and
cytosine Cytosine () (symbol C or Cyt) is one of the four nucleobases found in DNA and RNA, along with adenine, guanine, and thymine ( uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached ...
can be replaced by 5-methylcytosine. Some of these, such as pseudouridine and 5-methylcytosine, occur naturally in
eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...
s, while m1Ψ occurs naturally in archaea. Inclusion of these modified nucleosides alters the
secondary structure Protein secondary structure is the three dimensional form of ''local segments'' of proteins. The two most common secondary structural elements are alpha helices and beta sheets, though beta turns and omega loops occur as well. Secondary struct ...
of the mRNA, which can reduce recognition by the innate immune system while still allowing effective translation.


Significance of untranslated regions

A normal mRNA starts and ends with sections that do not code for amino acids of the actual protein. These sequences at the 5′ and 3′ ends of an mRNA strand are called
untranslated region In molecular genetics, an untranslated region (or UTR) refers to either of two sections, one on each side of a coding sequence on a strand of mRNA. If it is found on the 5' side, it is called the 5' UTR (or leader sequence), or if it is f ...
s (UTRs). The two UTRs at their strand ends are essential for the stability of an mRNA and also of a modRNA as well as for the efficiency of translation, i.e. for the amount of protein produced. By selecting suitable UTRs during the synthesis of a modRNA, the production of the target protein in the target cells can be optimised.


Delivery

Various difficulties are involved in the introduction of modRNA into certain target cells. First, the modRNA must be protected from ribonucleases. This can be accomplished, for example, by wrapping it in liposomes. Such "packaging" can also help to ensure that the modRNA is absorbed into the target cells. This is useful, for example, when used in
vaccine A vaccine is a biological preparation that provides active acquired immunity to a particular infectious or malignant disease. The safety and effectiveness of vaccines has been widely studied and verified. ...
s, as nanoparticles are taken up by
dendritic cell Dendritic cells (DCs) are antigen-presenting cells (also known as ''accessory cells'') of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. ...
s and macrophages, both of which play an important role in activating the immune system. Furthermore, it may be desirable that the modRNA applied is introduced into specific body cells. This is the case, for example, if heart muscle cells are to be stimulated to multiply. In this case, the packaged modRNA can be injected directly into an
artery An artery (plural arteries) () is a blood vessel in humans and most animals that takes blood away from the heart to one or more parts of the body (tissues, lungs, brain etc.). Most arteries carry oxygenated blood; the two exceptions are the pu ...
such as a coronary artery.


Applications

An important field of application are mRNA vaccines. Replacing uridine with pseudouridine to evade the innate immune system was pioneered by Karikó and Weissman in 2005. Another milestone was achieved by demonstrating the life-saving efficacy of nucleoside modified mRNA in a mouse model of a lethal lung disease by the team of Kormann and others in 2011. N1-methyl-pseudouridine was used in vaccine trials against Zika, HIV-1,
influenza Influenza, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptom ...
, and Ebola in 2017–2018. The first authorized for use in humans were COVID-19 vaccines to address
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) is a strain of coronavirus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the ongoing COVID-19 pandemic. The virus previously had a No ...
. Examples of COVID-19 vaccines using modRNA include those developed by the cooperation of BioNTech/
Pfizer Pfizer Inc. ( ) is an American multinational pharmaceutical and biotechnology corporation headquartered on 42nd Street in Manhattan, New York City. The company was established in 1849 in New York by two German entrepreneurs, Charles Pfize ...
( BNT162b2), and by Moderna ( mRNA-1273). The '' zorecimeran'' vaccine developed by
Curevac CureVac N.V. is a German biopharmaceutical company that develops therapies based on messenger RNA (mRNA). Legally domiciled in the Netherlands and headquartered in Tübingen, Germany, the company was founded in 2000 by Ingmar Hoerr (CEO), S ...
, however, uses unmodified RNA, instead relying on codon optimization to minimize the presence of uridine. This vaccine is less effective, however. Other possible uses of modRNA include the regeneration of damaged heart muscle tissue and cancer therapy.


References


Further reading

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