Optimizing mRNA for transformative drug discovery

by Dr. Alexander Zehnder, CEO, CureVac

The success of mRNA vaccines in COVID-19 propelled the role of this new modality to the forefront of medicine. Not only are new prophylactic mRNA vaccines for a variety of respiratory diseases advancing through clinical development, but mRNA drugs are now being developed in many broad applications, including cancer and rare diseases.

The extraordinary therapeutic potential of mRNA lies in its role as an information-carrying molecule that directs the production of proteins in every living cell. Ultimately, mRNA therapy allows the body to make its own drugs by mimicking endogenous mRNAs. But what does it take to design a potent and successful mRNA vaccine or therapy?

Challenges of using mRNA as medicine

Using mRNA as medicine has long been of interest given its potential to overcome the limitations of existing treatment modalities. However, mRNA has historically been limited by several theoretical and practical hurdles, namely:

• Stability: Naked mRNAs are rapidly degraded by RNase enzymes present throughout the environment, limiting the ability to generate therapeutically stable mRNAs. In addition, the endogenous mRNA degradation pathways in the body limit the duration of their therapeutic effect. Effective mRNA treatments need to be shielded from these enzymes both as drug formulations and when introduced into the body.
• Absorption by cells: Although it may be possible to deliver mRNA directly to target tissues without a delivery system in certain cases, the presence of RNA-degrading enzymes in blood and interstitial fluid rapidly converts any extracellular mRNA. More effective mRNA-based drugs require delivery systems that deliver mRNA efficiently into cells.
• expression level: The protein expression levels of synthetic mRNAs obtained by in vitro production have historically been considered too low for therapeutic purposes. This problem could not be overcome until the structural features of the mRNA were developed to significantly increase mRNA expression.
• Immunogenicity: mRNA has the ability to activate receptors on cells strongly as well as sensors within cells. This process triggers an innate immune response and can lead to the death of protein translation in cells. Effective mRNA-based drugs need to modulate the immune system according to the indications for the disease they are targeting.

The potency of a given mRNA drug product is a combination of the efficacy potential of the mRNA encoding the antigen protein and the delivery system that transports the mRNA to the cell. The success of mRNA therapy and vaccines can only be achieved if the several components are optimized to work together efficiently. These components include the mRNA backbone, the corresponding antigen, and the delivery system that encapsulates the mRNA, such as the lipid nanoparticle (LNP) system.

the mRNA backbone – the foundation

Some applications may require the highest possible protein expression but only for a limited time, for other applications long-lasting protein expression may be key. The peak level and duration of protein expression can be adjusted by the choice or design of enhancer and stabilizer elements in the untranslated region of the mRNA, or the secondary structure of the mRNA molecule. Another example is the optimization of how ORF instructs the synthesis of proteins encoded by ribosomes. ORF sequences can be optimized to increase mRNA stability. The important thing is, it can
achieved without changing the sequence of the protein encoded by ORF. For example, at CureVac, our team found that by increasing the content of G and C nucleotides, mRNA stability and protein expression could be significantly improved, so we patented a strategy that replaces the A or U nucleotide at the third position in an open reading frame with a G or C nucleotide. The optimization strategy allowed us to develop a new mRNA backbone used in CV2CoV, CureVac’s second generation COVID-19 vaccine, to elicit a much greater humoral and cellular immune response and provide much better protective efficacy against SARS-CoV-2 challenge than with the first generation of vaccines in macaques.

The principle behind mRNA treatment is to use synthetic mRNA molecules to direct the production of proteins that will produce the desired immune response. The synthetic mRNA strand also contains a number of structural elements that characterize each natural mRNA: the 5′-cap, the 5′-untranslated region (5’UTR), the coding region, or open reading frame (ORF), and the 3′-untranslated region ( 3′ UTR) including the poly(A) tail.

Optimization of the mRNA backbone targets the creation of the most efficacious mRNAs for specific targets and indications by optimizing translation, stability and immunogenicity. Each of these parameters can be modified by changing the individual mRNA elements and their interactions are guided by the application envisioned.

For example, UTR contributes conclusively to the potential efficacy of mRNA drugs. Depending on the target and the indication, the pharmacokinetics of the required protein expression may differ.

Some applications may require the highest possible protein expression but only for a limited time, for other applications long-lasting protein expression may be key. The peak level and duration of protein expression can be adjusted by the choice or design of enhancer and stabilizer elements in the untranslated region of the mRNA, or the secondary structure of the mRNA molecule. Another example is the optimization of how ORF instructs the synthesis of proteins encoded by ribosomes. ORF sequences can be optimized to increase mRNA stability. Importantly, this can be achieved without altering the protein sequence encoded by ORF.

For example, at CureVac, our team found that by increasing the content of G and C nucleotides, mRNA stability and protein expression could be significantly improved, so we patented a strategy that replaces the A or U nucleotide at the third position in an open reading frame with a G or C nucleotide. The optimization strategy allowed us to develop a new mRNA backbone used in CV2CoV, CureVac’s second generation COVID-19 vaccine, to elicit a much greater humoral and cellular immune response and provide significantly better protective efficacy against SARS-CoV-2 challenges. compared to first-generation vaccines in macaques.

Each mRNA element together in combination with the overall sequence affects the degree of activation of the immune system by a particular mRNA. Therefore, the design and optimization of an mRNA backbone always considers multiple factors as well as the overall construct.

Protein – immune response stimulator

The next key component is the protein, or rather the instructions for encoding and producing the desired protein. Protein is recognized as foreign, so the body mounts an immune response to remove it. For example, the COVID-19 vaccine triggers an immune response to the spike protein on the surface of the SARS-Cov-2 virus.

Finding the right target plays a very important role in the development of cancer mRNA vaccines. Although tumor cells originate from normal cells, they contain unique molecules expressed on the cell surface, often mutated, that are involved in inducing cells to become cancerous. These molecules are recognized as foreign by the immune system, which can then mount an immune response against the tumor. The hard part is identifying the right protein to differentiate healthy cells from cells that develop into cancer.

Conventional target discovery focuses on mutations in the tumor exome, which is the coding portion of the genome but represents only about 1.5% of the total genetic information. At CureVac, we take a different approach. We performed whole genome sequencing and combined it with short and long RNA sequencing. In this way, complete genomic information from the tumor can be mapped.

Downstream of sequencing, bioinformatics integrates all the data to pick up precise changes in the DNA of tumor cells compared to healthy cells. Correlation of these data results in the identification of novel and potentially antigenic tumor proteins that can be used for cancer vaccine development.

modified versus unmodified mRNA

There has been much debate about the use of modified and unmodified mRNAs and their subsequent successful applications, especially for COVID-19 vaccines. The data obtained by BioNTech and Moderna for their COVID-19 vaccine is exciting and subsequent work by us and others in the field shows that the modified mRNA allows higher doses and thus higher levels of neutralizing antibodies – an important characteristic of vaccines. prophylaxis. Modified mRNAs have also shown clinical promise in cancer vaccine development. However, clinical trials of both modified and unmodified mRNA vaccines are ongoing to also determine the best induction of robust T-cell responses. This is an important aspect for cancer applications.

LNP – delivery vehicle

Lastly, LNP, is the most common and most clinically sophisticated delivery system used for mRNA products. Encapsulation of the mRNA strand within the LNP allows efficient delivery to the site of action within the cell without degradation, rapid excretion and hepatic clearance. This allows for higher bioavailability and longer half-life.

LNP is composed of different lipids which combine with mRNA to form lipid nanoparticles. They mimic low-density lipoproteins, so they can be taken up by cellular transport pathways to deliver mRNA cargo to cells. At CureVac, we have developed, independently and with commercial partners, several LNP technologies. The most important finding in our LNP study is that the choice of lipid, its composition, and concentration allows us to adjust for different immune responses. That is an important aspect to meet the specific requirements for different applications with bespoke LNP systems. For example, prophylactic vaccine requirements compared to cancer vaccines are very different.

LNP for vaccine prophylaxis especially should support strong antibody induction and minimized reactogenicity as most healthy individuals are
managed. Because prophylactic vaccines usually follow seasonal outbreaks, LNPs need to contribute to the overall stability of the vaccine for long-term storage in refrigerators or even room temperature. LNP systems for cancer vaccines on the other hand need to support the activation of signaling pathways in cells for robust induction of systemic immune responses in critically ill patients. Activation of certain cytokines and chemokines in the pathway may lead to higher reactogenicity but is critical for the induction of T-cell responses. For cancer vaccines, stability can be prioritized over stronger efficacy.

LNP studies have demonstrated a highly localized distribution within the immune compartment, favorable cellular and humoral immune responses in preclinical trials, as well as good initial room temperature stability data as dry presentation. Limited biodistribution to the immune compartment is essential for intramuscular injection to avoid unwanted antigen expression in distant organs such as liver, spleen and lung.

Conclusion

While much progress has been made in the field of mRNA, we need to continue to innovate so that the promise and full potential of mRNA can be realized. Even in the current science, vaccine prophylaxis and cancer fields, there are almost limitless possibilities for designing, optimizing and delivering new synthetic mRNA drugs.

The future looks bright for the sector, but especially for the patients who will one day benefit.