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A path out of the pandemic

In record time people around the world had access to an effective vaccine against the new deadly and dangerous coronavirus. The basic research discoveries made by Katalin Karikó and Drew Weissman paved the way for this historic achievement and have also led to a novel therapeutic technology – with great potential for many different diseases.

The Nobel Prize in Physiology or Medicine will this year be awarded to Katalin Karikó and Drew Weissman for their discoveries concerning “nucleoside base modifications that enabled the development of effective mRNA vaccines against COVID-19.”

So first of all, what are nucleosides and nucleoside base modifications? A nucleoside is composed of a sugar moiety and nucleobase, whereas a nucleotide is composed of a sugar, nucleobase, and at least one phosphate (or phosphate-like) group. Nucleosides play a key role in biology processes. They are involved in the retention, replication and transcription of gene information. In the most important nucleosides, the sugar is either ribose (RNA) or deoxyribose (DNA), and the nitrogen-containing compound is either a pyrimidine (cytosine, thymine (DNA), or uracil (RNA)) or a purine (adenine or guanine).

In nucleoside-modified messenger RNA (mRNA) some nucleosides have been replaced by other naturally modified nucleosides or by synthetic nucleoside analogues. These modRNA can be used to induce the production of a desired protein in certain cells.

A novel therapeutic technology

RNA was first injected into an animal with the idea of using it as a therapeutic in 1990, but it did not go anywhere. It was not until Karikó and Weissman started working on RNA, after a chance meeting in the late 1990s, and figured out why it was so inflammatory (and how to make it non-inflammatory) that the field became relevant and interesting, as Weissman described in an interview with Penn Medicine in 2020. In short, they discovered how to modify mRNA in a way that boosts protein production while minimizing harmful inflammatory responses. Their key discovery, that mRNA could be altered and delivered effectively into the body to activate the body’s immune system, was published in 2005 (Karikó et al., Immunity, 2005).

Their findings have fundamentally changed our understanding of how mRNA interacts with our immune system.”

Their discoveries have led to a novel therapeutic technology, including the rapid development of mRNA-based vaccines that elicit a robust immune response, including high levels of antibodies that attack a specific infectious disease that has not previously been encountered.

“Their findings have fundamentally changed our understanding of how mRNA interacts with our immune system and the Laureates contributed to the unprecedented rate of vaccine development during one of the greatest threats to human health in modern times,” wrote the Nobel Assembly at Karolinska Institutet at the time of the announcement.

 

Spike production following mRNA vaccination and recognition of spike by B cells. Following uptake of mRNA into cells, facilitated by lipid nanoparticles, the mRNA acts as a template for spike protein production. Spike is the transiently expressed on the cell surface, where it is recognized by B cells via their B cell receptors (BCRs), stimulating the secretion of spike-specific antibodies. Illustration: Mattias Karlén

 

A COVID-19 vaccine in record time

At an early stage of the COVID-19 pandemic, both Pfizer/BioNTech and Moderna utilized Karikó and Weissman’s nucleoside-modified mRNA technology (and other mRNA vaccine-related improvements) to develop their COVID-19 vaccines. What usually takes 10-20 years to develop was now developed in record time and to date, hundreds of millions of people all over the world have received mRNA vaccines against the coronavirus.

Unlike other types of vaccines, a live or attenuated virus is not injected or required at any point with mRNA vaccines. Since RNA is the producer of proteins you can make an RNA that codes for a protein, and for COVID-19 this was the spike protein found on the virus. When you inject the RNA that codes for the spike protein, the cells will take it up and produce it in large quantities. The advantage of using RNA is that each RNA can make 1,000 to 100,000 proteins. In addition, RNA itself is a very rapid platform and you only need the sequence to start producing a vaccine, described Weissman in the interview with Penn Medicine in 2020.

In addition, the mRNA in the vaccine candidate is produced synthetically and means it can be created much faster than has been done for past vaccines.”

“The mRNA vaccines are promising because of their potential for high potency and ability to boost immune responses, engaging several arms of the immune system, such as antibody-producing B cells and anti-viral T-cells,” explained Michael Dolsten, CSO of Pfizer, in a previous interview with NLS. “This is different from adenovirus-based vaccines, which traditionally can only be given as a single, likely short-lasting administration, and protein-based vaccines that usually mainly give rise to protective antibodies and less to T-cell immunity. In addition, the mRNA in the vaccine candidate is produced synthetically and means it can be created much faster than has been done for past vaccines.”

 

Novel Corona-virus SARS-CoV-2 Spike Protein. Illustration: NIAID

 

What’s next?

Building on Karikó and Weismann’s groundbreaking work, researchers are now developing modified mRNA therapies within a number of different areas, including cancer, infectious disease and autoimmune disorders. Before COVID-19 hit, Weissman and Karikó had also already set up clinical trials for mRNA vaccines for genital herpes, influenza, and HIV, and the clinical potential extends beyond vaccines to other advanced therapies, such as protein replacement, gene therapy, and cancer immunotherapy.

Within cancer the difference, compared to the COVID-19 vaccine which is prophylactic and protects people from a virus, is that a cancer mRNA vaccine is an intervention, a treatment given to patients with the aim that their immune systems would be activated in a way that would attack tumor cells. Recently Moderna and Merck announced that when used together with Merck’s cancer immunotherapy, Keytruda, their mRNA cancer vaccine reduced the risk of certain skin cancers from returning and patient deaths by 44% (compared with Keytruda alone). The British government also announced that it was partnering with BioNTech to enroll as many as 10,000 patients in trials of a new mRNA cancer vaccine.

The biggest challenge in developing these types of mRNA vaccines for cancer, though, is just how personal it has to be.

We could see a scenario where patients are put on an off-the-shelf vaccine immediately while a personalized vaccine is being produced, if the patient harbors the antigens in an available off-the-shelf cancer vaccine.”

“Personalized cancer vaccines can be tailor-made to each patient, but will always need to undergo manufacturing before administration and therefore take a bit longer before being administered to the patient. Universal cancer vaccines, or off-the-shelf cancer vaccines, have the benefit of immediate administration to the patient, but they require the patient to harbor specific antigen targets. We could see a scenario where patients are put on an off-the-shelf vaccine immediately while a personalized vaccine is being produced, if the patient harbors the antigens in an available off-the-shelf cancer vaccine,” described Michael Engsig, CEO of Nykode Therapeutics, in a previous interview with NLS (2023).

Development & manufacturing of mRNA

Due to the great progress made during the COVID-19 pandemic, mRNAs are a fast-emerging class of biotherapeutics. These therapies offer a new opportunity for targeted treatment of challenging diseases and flexible manufacturing. However, mRNA is a still-young process modality with diverse challenges. NLS asked Dr. Maya Fuerstenau-Sharp, Head of Marketing, Cell Culture Technologies, Bioprocess Solutions, at Sartorius about her experiences of mRNA development and manufacturing, and her view on the field and its potential.

The potential of mRNA is broad, she says, “mRNA is highly multivalent, making it a good candidate for targets with high variation, such as combining strains for COVID and influenza or generating effective cancer vaccines.”

Early studies have for example demonstrated the ability for in vivo mRNA delivery to create CAR-T cells to treat cancer and heart disease. She says, “mRNA can also turn cells in the body into factories for functional proteins or antibodies. Altogether, mRNA is a powerful new therapeutic class.”

Fuerstenau-Sharp and her colleagues at Sartorius offer equipment for mRNA therapeutics process development, manufacturing and analysis. They have created new analytical chromatography tools to address the developing analytical methods for both development and process characterization. “We have published data on generating GMP mRNA in volumes as low as 100mL in our rocking motion bioreactor, which is useful for indications like cancer vaccines that require very low process volumes. These types of activities will enable our customers to unleash the full potential of mRNA from personalized medicines to global-scale vaccinations,” she describes.

Challenges for mRNA development and manufacturing include the cost of raw materials, the yield of reactions that produce mRNA, and scale of manufacturing processes to meet emerging indications.”

Challenges for mRNA development and manufacturing include the cost of raw materials, the yield of reactions that produce mRNA, and scale of manufacturing processes to meet emerging indications, explains Fuerstenau-Sharp. “In vitro transcription reactions have expensive enzymes and reagents, which cause the cost per volume to be drastically higher than traditional biological manufacturing. This high raw material cost means that development work done in traditional bioprocessing equipment, which is comparatively oversized, is highly expensive,” she says.

 

Left: mRNA production at BioNTech, Marburg. Right: Maya Fuerstenau-Sharp, Head of Marketing, Cell Culture Technologies, Bioprocess Solutions, Sartorius.

 

Together with her colleagues she has been working hard to create publicly accessible methods to increase productivity, lower the scale of GMP manufacturing and decrease the cost of goods necessary to produce IVT batches. “Additionally, stability and tissue-specific targeting remain areas for improvement and we have launched a set of novel cationic lipid nanoparticles as a step towards addressing these challenges,” describes Fuerstenau-Sharp.

New RNA classes, such as self-amplifying (saRNA) and circular (circRNA), have distinct advantages over traditional mRNA.”

Current trends within mRNA development and manufacturing include new indications, new RNA sub-classes and smaller process volumes. The multivalency of mRNA makes it highly suitable for personalized medicine like cancer vaccines, believes Fuerstenau-Sharp. “New RNA classes, such as self-amplifying (saRNA) and circular (circRNA), have distinct advantages over traditional mRNA. The saRNA is more efficient and requires a lower dosage to achieve the same effect. The circRNA is less sensitive to degradation and has a longer-term expression profile in the body. These new technologies require significantly smaller process volumes than traditional biopharma due to the nature of personalized indications and highly efficient RNA molecules,” she says.

Finally I ask Maya Fuerstenau-Sharp what areas she thinks will benefit next from the nucleoside-modified mRNA technology.

“Nucleoside-modified mRNA elicits a reduced immune response while increasing target protein production. This makes it a promising candidate for continued adoption across all mRNA fields, including vaccines, cancer therapy, and regenerative medicine,” Fuerstenau-Sharp concludes.

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