What is the future of mRNA based therapeutics, and their main challenges?

mRNA is a ribonucleic acid used by cells as an intermediate carrier of the genetic information contained in the DNA to synthesize proteins, and corresponds to the copy of one of the two strands of DNA. Over the past years, major technological innovation has allowed mRNA to become an increasingly used tool for therapeutic purposes. The recent Covid-19 epidemic has demonstrated the effectiveness of mRNA vaccines against infectious diseases. Indeed, these are faster to develop, simpler, cheaper to manufacture, and pose fewer health risks compared to conventional vaccine approaches 1, 2, 3, 4. In addition, the use of mRNA is further being considered as a therapeutical solution against non-infectious diseases such as cancer5, to produce chimeric antigen receptor T cells (CAR-T) used in immunotherapy, as well as for therapies based on the administration of proteins or antibodies.

Despite recent technological advances in terms of mRNA modifications and delivery solutions, several important challenges remain for mRNA-based therapeutics to be effective: mRNA Immunogenicity and stability, improvement of the transcription and translation rate, level of antigen production.

In this article we will discuss the future of mRNA vaccines, the importance of mRNA sequence optimization to maximize the impact of production, delivery, and administration of therapeutic mRNAs, and how Tebubio’s research grade mRNA production service can support you in responding to your challenges and enable you to quickly advance your projects to the next steps.

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The importance of UTRs for high translation efficiency

mRNA comprises 3 regions: a 5’ untranslated region (5’-UTR), a coding region for a protein (CDS), and a 3’ untranslated region (3’-UTR). The 5’-UTR region contains the translation signals allowing the recruitment of the ribosome for the translation of the coding region into protein. A cap structure, a highly methylated modification at the 5’ end of the RNA, protects it from degradation, marking it as “self” to avoid recognition by the innate immune system. At the 3’ end, poly(A) tail and additional elements make the RNA molecule more stable and prevent its degradation.

The amount of protein produced from any given mRNA depends on its in-cell translational rate (how well the translational machinery initiates and elongates the CDS) as well as of the mRNA half-life (how long it remains intact inside cells). The 5’- and 3’-UTRs flanking the coding sequence profoundly influence mRNA translation and stability. In particular, they are the elements with the greater impact on ribosome load, which corresponds to the average number of ribosomes associated to a given mRNA6.

Historically, only a handful of natural UTR elements, derived from either cellular or viral genomes, have been tested and characterized. Among them are the 5’- and 3’-UTRs from human hemoglobin subunit beta (hHBB), which is one of the most efficiently expressed mammalian mRNAs and is commonly used in studies on mRNA translation and stability. Viruses have evolved regulatory elements to take over the host translation machinery and effectively promote translation of their own mRNAs. For example, internal ribosome entry sites (IRESs) can recruit ribosomes to initiate translation without the need for the eukaryotic initiation factors.

More recent work has focused on systematic analysis of libraries of natural, mutated or rationally designed UTRs for their potential to enhance or fine-tune mRNA translation or stability6.

Tebubio has developed a proprietary pDNA template with optimized 5’- and 3’ -UTRs to ensure optimal transcription and translation of the target protein in mammalian cells. Expression tests in mammalian cells (fig.1) show that the protein expression level of the eGFP mRNA produced with our proprietary vector, is comparable to the eGFP mRNA prepared byTriLink, our CDMO partner and leader in mRNA production, and superior to that developed by a competitor.

Tebubio, TriLink, and Competitor GFP mRNA
Figure 1 – Comparison of eGFP protein expression in HEK293 by fluorescence microscopy using FITC (510 nm) filter, after transfection with mRNA produced by Tebubio, TriLink Biotechnologies, or a competitor mRNA supplier

In addition, in our plasmid, the poly-A tail has also been optimized to guarantee you a constant tail length, for better mRNA stability, and batch-to-batch production reproducibility.

The role of secondary structure in transcription and translation efficiency

Besides the nature of the UTRs, both translational efficiency and half-life are influenced by multiple factors in primary nucleotide sequence, including GC content, codon usage, codon pairs, and secondary structure. While a low secondary structure in the 5’-UTR and in the first ~10 codons of the CDS is required for high translation efficiency, mRNAs containing highly structured CDS and 3’-UTR have been found to give the best protein output6. This is due to the longer half-life of highly structured mRNA. Indeed, mRNA length and structure are the main drivers of in-cell and in-solution mRNA stability.

Whereas highly structured mRNAs can be efficiently translated thanks to the strong intrinsic helicase activity of the ribosome while translocating along mRNA, secondary structures may pose significant issues to the transcription efficiency. Indeed, during in vitro transcription, the RNA polymerase can be slowed down or even blocked by sequences rich in G and C and hairpin-shaped secondary structures, leading to low yields of production or to the generation of truncated forms of RNA.

For this reason, Tebubio proposes a series of optimizations in order to maximize the transcription efficiency whilst ensuring high protein expression in the species of choice.

First, with a codon optimization of the CDS that includes a reduction of the G and C content (fig.2) as well as of the secondary structures such as hairpins.

GC Content Analysis

Plot of GC content of wild-type gene and optimized gene

Hairpin analysis of Wild-type and Optimized gene
Figure 2 – GC content and hairpin analysis in an example sequence, before (blue) and after (red) codon optimization

Some sequences, especially those of several thousands of nucleotides, are of such complexity that they cannot be sufficiently improved only by codon optimization. For difficult-to-transcribe sequences, the production and purification conditions can be tuned to increase mRNA quality. For example, the interactions at the origin of the secondary structures are based on hydrogen bonds, which can be weakened by modifying the temperature of the transcription reaction.

Moreover, RNA purification with silica gel is based on the ability of the latter to absorb nucleic acids reversibly depending on the salt concentration, pH, and temperature. Modulating one or more parameters, based on the complexity of your sequence often results in fewer truncated forms and better homogeneity of your mRNA produced (fig.3).

Post Purification RNA analysis by Agilent Tape Station 4150 - before and after GC optimization
Figure 3 – mRNA size and homogeneity analysis, by tape station, of difficult-to-transcribe sequences before and after optimization.
Presence of 2 spikes before optimization (mRNA truncated forms) vs. a unique and homogeneous spike and mRNA produced after optimization

Reducing immunogenicity and improving half-life by uridine depletion and modification

Potential immunogenicity of mRNA transcripts is a major issue for some mRNA-based medicines. Indeed, unmodified exogenous mRNA activates Toll-like receptors (TLRs), resulting in upregulation of proinflammatory cytokines such as IFN-I, IL-6, IL-12, TNF-α and chemokines, which hamper mRNA translation through eIF2 activation by protein kinase R (PKR) and promote RNA degradation8, 9. To overcome this, therapeutic mRNA can be modified by replacing nucleotides with analogues that make it less immunogenic and more resistant to degradation (fig. 5). For example, the first mRNA vaccines to be approved, BNT162b2 and mRNA-1273 against SARS-CoV-2, have all uridines replaced by N1-methylpseudouridine, a naturally occurring analogue of uridine present in various types of RNA in eukaryotic cells.

Intracellular pathways of innate immune response in cells transfected with non-modified (left) versus chemically modified exogenous mRNA (right)
Figure 4  – Intracellular pathways of innate immune response in cells transfected with non-modified (left) versus chemically modified exogenous mRNA (right) (Moradian et al, 20229)

In addition to reducing the immunogenicity, the replacement of uridine with pseudouridine has been found to stabilize mRNA in-solution6, 7. Indeed, the RNA linkages 5’ of uridine residues are particularly susceptible to degradation, which can be alleviated through the inclusion of uridine analogues.

To both limit immune sensing and enhance protein production, Tebubio proposes codon optimization that includes uridine depletion. Moreover, the remaining U in the mRNA sequence, as well as other nucleotides, can be replaced with modified analogs. While the best modifications depend on your cell type, N1-methylpseudouridine is the most used, as it provides a high protein expression in many cellular systems (fig.6).

Comparison of eGFP expression levels lin 6 cell types
Figure 5 – Comparison of eGFP expression levels lin 6 cell types, using unmodified or modified mRNA with different nucleotide analogues (TriLink Biotechnologies)

Self-amplifying RNA – the future of RNA vaccines

saRNA is an mRNA molecule that codes for four additional proteins in addition to the antigen of interest. These four additional proteins are non-structural viral proteins encoding a replicase, which is able to amplify the same saRNA inside cells. As it does not contain viral structural protein, it is unable to produce infectious virus. Since the time that the mRNA remains functional inside the cells is a major driver of the corresponding protein expression, self-amplifying RNA (saRNA) is regarded as the solution to induce the production of proteins over long periods of time from a single dose of RNA (fig.6). With mRNA vaccines currently on the market, expression of the antigen of interest is proportional to the number of mRNA molecules successfully delivered into the cells. Achieving a sufficient level of expression for protection or immunomodulation may thus require high doses or repeated administrations, as in the case of vaccines against SARS-COVID-19. Vaccines based on saRNA could remedy this limitation, enabling reducing of the dose of RNA to be administered, with consequent benefits in terms of cost and speed of production, as well as a reduction in side effects. In a pandemic context, a saRNA vaccine would allow the production of 10 to 1,000 times more doses compared to an mRNA vaccine (Blakney, Vaccine Strategies, 2021).

Figure 6 - the difference between mRNA and saRNA (Blakney ET AL; 2021 (13))
Figure 6 – the difference between mRNA and saRNA (Blakney et al.; 202113)

The saRNAs currently under development are derived from alphaviruses, such as the Venezuelan equine encephalitis virus (VEEV), the Semliki forest virus (SFV) or the Sindbis virus. A first phase I/II clinical trial was carried out in 2020 for a saRNA vaccine against SARS-COVID-19 and others are in development for infectious diseases such as influenza (influenza virus), rabies (rabies virus), malaria (Plasmodium parasite), chlamydia (chlamydia trachomatis bacteria), viruses such as HIV-1, Ebola, RSV and Zika, as well as for oncology applications such as melanoma and colon carcinoma10. Two different approaches are currently being studied for the generation of saRNA therapeutics:

1 – Use of a single DNA template containing the gene of interest and those of the four replicase-coding proteins (Fig.7B).

2 – Use of 2 DNA templates containing respectively the gene of interest and the four additional proteins (Fig.7C).

Different saRNA approaches vs conventional mRNA
Figure 7 – Figure showing different saRNA approaches vs conventional mRNA (Schmidt et al, 2023(11))

A recent study12 has demonstrated a double interest in the second approach. On one hand, the separation of the system into 2 RNAs simplifies its delivery into the cells, since the saRNA coding for all the proteins is difficult to deliver because of its very large size (~ 10,000 nt). On the other hand, it would make it possible to vary only the DNA matrix coding for the gene of interest without having to modify the replicase matrix. Moreover, the RNA coding for the replicase could be combined with several RNAs coding for different antigens, making it possible to generate multivalent RNA vaccines. At Tebubio, our project managers are already working on the production of saRNA and on the optimization of the best approach based on researchers needs.

Conclusion

Even if the use of mRNA as a therapeutic solution has proved it efficacy, numerous challenges remain to be overcome in order to improve the efficiency of mRNA in the treatment of many diseases.
At Tebubio, with our mRNA production service, our goal is to facilitate your research by quickly providing optimised research grade sequences (μg production in as little as 5 days), to enable you to validate your PoCs and to advance your research projects as quickly as possible.

To learn more about our mRNA production service and our optimization capacities, we invite you to read our latest Application Note and to visit our webpage dedicated to our small scale mRNA services.

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References

1. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines – a new era in vaccinology. Nat Rev Drug Discov. 2018

Apr;17(4):261-279. doi: 10.1038/nrd.2017.243. Epub 2018 Jan 12. PMID: 29326426; PMCID: PMC5906799.

2. Teo SP. Review of COVID-19 mRNA Vaccines: BNT162b2 and mRNA-1273. J Pharm Pract. 2022 Dec;35(6):947-951. doi:

10.1177/08971900211009650. Epub 2021 Apr 12. PMID: 33840294.

3. Pollard AJ, Bijker EM. A guide to vaccinology: from basic principles to new developments. Nat Rev Immunol. 2021

Feb;21(2):83-100. doi: 10.1038/s41577-020-00479-7. Epub 2020 Dec 22. Erratum in: Nat Rev Immunol. 2021 Jan 5;: PMID:

33353987; PMCID: PMC7754704.

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PMC9108694.

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GC, Wellington-Oguri R, Fujii K, Sharma E, Watkins AM, Nicol JJ, Romano J, Tunguz B, Diaz F, Cai H, Guo P, Wu J, Meng

F, Shi S, Participants E, Dormitzer PR, Solórzano A, Barna M, Das R. Combinatorial optimization of mRNA structure,

stability, and translation for RNA-based therapeutics. Nat Commun. 2022 Mar 22;13(1):1536. doi: 10.1038/s41467-022-

28776-w. PMID: 35318324

7. Mauger DM, Cabral BJ, Presnyak V, Su SV, Reid DW, Goodman B, Link K, Khatwani N, Reynders J, Moore MJ, McFadyen

IJ. mRNA structure regulates protein expression through changes in functional half-life. Proc Natl Acad Sci U S A. 2019

Nov 26;116(48):24075-24083. doi: 10.1073/pnas.1908052116. Epub 2019 Nov 11. PMID: 31712433

8. Karikó K. Modified uridines are the key to a successful message. Nat Rev Immunol. 2021 Oct;21(10):619. doi: 10.1038/

s41577-021-00608-w. PMID: 34580453

9. Moradian H, Roch T, Anthofer L, Lendlein A, Gossen M. Chemical modification of uridine modulates mRNA-mediated

proinflammatory and antiviral response in primary human macrophages. Mol Ther Nucleic Acids. 2022 Jan 10;27:854-

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10. Blakney AK. The next generation of RNA vaccines: self-amplifying RNA. Vaccine Strategies 2021

11. Schmidt C, Schnierle BS. Self-Amplifying RNA Vaccine Candidates: Alternative Platforms for mRNA Vaccine

Development. Pathogens 2023, 12, 138. https:// doi.org/10.3390/pathogens12010138

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Splitzicons. Front Mol Biosci. 2018 Jul 26;5:71. doi: 10.3389/fmolb.2018.00071. eCollection 2018. PMID: 30094239

 

For any further questions, please contact Tebubio’s Project Managers, we’ll be pleased to help.

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