Nucleoside-modified messenger RNA

A nucleoside-modified messenger RNA (modRNA) s a synthetic, chemically modified messenger RNA (mRNA) in which individual nucleosides are replaced by other naturally modified nucleosides or by synthetic nucleoside analogues. modRNA is used experimentally or therapeutically to induce the production of a desired protein in certain cells.

Prerequisites

In a cell, messenger RNA is produced by synthesising a ribonucleic acid (RNA) strand according to a deoxyribonucleic acid (DNA) template, with the codogenic strand section serving as a matrix. This process is known as transcription. The mRNA is then read from ribosomes, which in turn serve as a blueprint for the synthesis of proteins by specifying their protein primary structure. The process of protein biosynthesis described last is called translation.

Principle of nucleoside modification

To induce cells to synthesise proteins that they do not normally produce, it is possible to use modRNA, in whose nucleotide sequence the amino acid sequence of these proteins is encoded. The mRNA synthesised in vitro must then be introduced into the organism, for example injected, taken up into the target cells and read off there. In this way, translation takes place without prior transcription. In other words, a blueprint for foreign proteins is smuggled into the cells. To achieve this goal, however, one must bypass systems that are used in the human organism to prevent the penetration and translation of foreign mRNA. Firstly, there are enzymes (ribonucleases) that break down "normal", i.e. unmodified mRNA. On the other hand, there are also intracellular barriers against foreign mRNA. When single-stranded RNA (ssRNA) is absorbed into endosomes via the cell membrane, it is recognised by the toll-like receptors 7 and 8, which belong to the innate immune system. This ultimately leads to the shutdown of protein synthesis in the cell, the release of interferons and cytokines and the activation of the transcription factors TNF-alpha and AP-1, which can lead to programmed cell death (apoptosis). This can be circumvented by modifying the system for in vitro production of mRNA in such a way that the similar (also naturally occurring) pseudouridine (Ψ) or N1-methyl-pseudouridine (m1Ψ) is incorporated instead of the physiological nucleoside uridine or 5-methylcytosine instead of cytosine. N1-methyl pseudouridine and 5-methylcytosine do not occur naturally. If an mRNA contains one or two of these modified nucleosides, this leads to a change in the secondary structure, which on the one hand prevents it from being recognised by the innate immune system, but on the other hand still allows effective translation to a protein.

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 3' and 5′ ends of an mRNA strand are called untranslated regions (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.

Obstacles, use of nanoparticles

To introduce modRNA into certain target cells, you face various difficulties. Firstly, the modRNA must be protected from ribonucleases. This can be done, for example, by wrapping it in lipid nanoparticles (solid lipid nanoparticles). Such "packaging" can also help to ensure that the modRNA is absorbed into the target cells. This is useful, for example, when used in vaccines, as nanoparticles are taken up by dendritic cells and macrophages, both of which play an important role in activating the immune system.

Furthermore, it may be desirable that the modRNA applied is specifically introduced into certain 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 intra-arterially into the coronary arteries, for example.

Risks

If the modRNA does not reach the target cells but other cells, undesired effects can occur. For example, if the encoded protein is actually supposed to stimulate heart muscle cells to proliferate, but is mistakenly produced in other cells, this could lead to proliferations. However, such a negative effect is limited in time by the fact that, despite its increased stability compared to normal mRNA, the modRNA is ultimately degraded, as are the proteins it codes.

It was also argued that changes in the genome of the cells, i.e. mutations, could be triggered with consequences including the development of cancer. However, it should be remembered that the genetic information is present in the cell nucleus as DNA (not as RNA) and modRNA does not enter the cell nucleus. Furthermore, there is no reverse transcriptase in the human body, i.e. no enzyme that can transcribe mRNA into DNA. While there are viruses in humans that produce reverse transcriptases (for example HIV) and that these reverse transcriptases could lead to reverse transcription of modRNA, these reverse transcriptases of viruses are highly specific and only transcribe the virus' own RNA, so this problem can probably be neglected.

Uses

Currently, the most important field of application of modRNA is the production of vaccines against SARS-CoV-2. The vaccines developed by the cooperation of the companies BionNTech/Pfizer/Fosun Internationaland by Curevac and Moderna as well as other businesses as protection against COVID-19 disease use modRNA technology.

Other possible uses of modRNA include the regeneration of damaged heart muscle tissue and cancer therapy.

Further reading

• Kenneth R. Chien, Lior Zangi, Kathy O. Lui (2015-01-01), "Synthetic Chemically Modified mRNA (modRNA): Toward a New Technology Platform for Cardiovascular Biology and Medicine", Cold Spring Harbor Perspectives in Medicine, vol. 5, no. 1, pp. a014035, doi:10.1101/cshperspect.a014035, ISSN 2157-1422, PMC 4292072, PMID 25301935{{citation}}: CS1 maint: multiple names: authors list (link)

References

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