mRNA delivery: from bench to clinics

Image of a syringe.

Recent developments of mRNA vaccines to protect people from SARS-COV-2 are making headlines. The first two successful mRNA vaccines, the immunization jointly developed by Pfizer and BioNTech and a second vaccine developed by Moderna, are turning mRNA vaccines into a household topic. For many scientists, this is not only great news for public health but also a proof-of-concept that mRNA vaccines can work! Today, I'll share with you some significant scientific discoveries and principles that make mRNA vaccines possible. 

What is mRNA?
mRNA is the abbreviation of Messenger RiboNucleic Acid. As the name suggests, it is responsible for relaying messages encoded in DNA molecules to protein production in cells. 

The synthesis of mRNA, a process called transcription, is carried out by RNA polymerase. tRNA molecules help to bring in the bricks needed for protein synthesis. For mRNA vaccine production, mRNA synthesis starts with a DNA template, and the mRNA molecules are synthesized using a phage RNA polymerase. 

How do mRNA vaccines work?
COVID mRNA vaccines function by delivering the mRNA that encodes spike proteins. Spike proteins are a protein on the surface of the SARS-COV-2 virus, and it plays an essential role in facilitating viruses to enter the cell. By delivering mRNA molecules that encode this protein into human bodies, cells inside the human body will synthesize this spike protein and prime the immune system accordingly. A diagram from Bloomberg news summarizes this well. 

How is mRNA delivered?
Most of the mRNA vaccines encapsulate mRNA molecules in lipid nanoparticles. Other delivery methods in RNA therapeutics include using polymeric nanoparticles comprising of RNA and cationic polymer. For small interfering RNA (siRNA), conjugating RNA to N-acetylgalactosamine, which targets receptors on hepatocytes for uptake, is also an option. 

What are the difficulties for mRNA vaccine development?
The hurdles for developing an effective mRNA vaccine can be divided into two parts: (1) mRNA cannot enter the cell intact, and (2) it cannot be translated into protein efficiently. 

• mRNA cannot enter the cell intact. 
  • Naked mRNA molecules are not stable. mRNA is easily degraded by nucleases. Thus, protecting these mRNA molecules is a hurdle that must be overcome. 
  • Naked mRNA cannot enter cells through free diffusion. mRNA molecules are large and negatively charged, meaning they cannot passively pass through cell membranes. 
• mRNA cannot be translated efficiently.
  • Naked, single-stranded RNA triggers cells' antiviral defense. Cells generate an immune response that results in active degradation of these mRNA molecules. Consequently, no protein-of-interest can be synthesized. 
  • Non-optimal codon usage (e.g., Use of rare codons or codons with low tRNA abundance). mRNA has a specific code wherein every three bases (drawn from a pool of A, U, C, G, and other modified bases) encodes an amino-acid needed for the protein. Strangely, some codes work better than others. Non-optimal choice of codes (base combinations) may result in bottlenecks in protein yield during translation. 
  • mRNA forms improper secondary structures. mRNAs can fold upon each other, causing secondary structures. Some secondary structures of mRNA hinder the binding of polymerase or other regulatory components that result in inefficient protein synthesis. 
  • Non-optimal regulatory regions (e.g., 5'UTR, 3'UTR, ployA tail). Non-optimized regions of mRNA that do not directly code for protein production may lead to insufficient binding of regulatory protein complexes that promote the translation of mRNA. 

What are the scientific discoveries that help to overcome these problems?
Most of the work falls into two categories: (1) direct mRNA modifications and (2) optimization of delivery particles. 

• Direct mRNA modification. 
  • Chemical modification of mRNA molecules. Most of the chemical modifications focus on either minimizing cells' antiviral defense or improving mRNA's stability. Modifications include capping mRNA at 5' end, modifying the sugar backbone of mRNA (e.g., methylate 2' hydroxyl group, add Amide-3 linkage), and modifying the mRNA (e.g., methylate cytidine, modify uridine to 5-Bromo-uridine). 
  • Codon optimization. mRNA codes can be changed to exclusively use codons with high tRNA abundance, and tRNAs expressed efficiently in a specific tissue. Codons can also be optimized to minimize pesky secondary structure formation. 
  • Regulatory region optimization can lead to improved binding of regulatory components to enhance protein translation and improve mRNA stability. No gold standard for this method has been developed yet. However, development in bioinformatics tools is improving this standard practice. 
• Optimization of delivery particles
  • Lipid nanoparticle engineering. Many efforts have been made to increase mRNA stability by adding lipids with positive charges (as mRNA are negatively charged) to improve cells' uptake of mRNA particles and promote endosome escape. 
  • Addition of chemicals in lipid nanoparticles. One example is to add MPLA, a chemical that impairs antiviral defense and promotes T-cell activation. 

What are the differences between the Pfizer and Moderna vaccines?
Both vaccines use lipid nanoparticles to deliver mRNA that encodes SARS-COV-2's spike proteins. However, because specific formulations are secret, the exact differences are unknown. Yet, it is speculated the differences in the lipid components are the reason why Pfizer's vaccine needs to be preserved at an ultra-cold temperature. In contrast, Moderna's vaccine can be shipped in a regular freezer. The difference in lipid composition may also explain why the Pfizer vaccine results in fewer side effects.

What else to look for?
Moderna's mRNA vaccines against other infectious diseases, such as its Zika vaccine currently in clinical trials, are worth watching. It posted encouraging Phase I clinical trial data and may come to the market sometime in the future. 

Other applications of mRNA vaccine technology include immunizations for cancer, such as a cancer vaccine jointly developed by Moderna and Merck that targets common mutations in KRAS, an oncogene that is often mutated in cancer patients. ​

Author

Yuezhe Li, @YuezheL
PhD candidate studying biomedical sciences at UCONN Health researching ciliopathy and diabetes. Also loves baking, reading, and traveling.