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Friday, April 09, 2021

SARS-CoV-2 mRNA vaccines: RNA + lipid nanoparticles

The new mRNA vaccines are the result of extensive research over the past thirty years or so. They are marvels of technological innovation but probably not just for the reasons you imagine. The basics of therapeutic mRNA synthesis have been around for about ten years but the problem was how to get the RNA into cells. That requires specialized lipid nanoparticles and making those has been the most recent technological advance. A lot of this research was done in Canada. I found a nice paper (Buschmann et al., 2021) that covers this research and I'll summarize the important points for those of you don't have time to read it.

The mRNA

Normal messenger RNA is susceptable to nuleases and is not readily taken up by human cells. In addition, it elicits an innate immune response that results in supression of translation through phosphorylation of eIF2a. The immune response can be blocked by incorporating modified nucleotides than are not recognized by the various receptors that stimulate the normal response. This was discovered over ten years ago. These modified nucleotides, such as N1-methylpseudouridine, were used to make the SAR-CoV-2 vaccine.

The mature vaccine RNA is produced by in vitro transcription from plasmid DNA and the product contains the coding region plus 5' and 3' UTRs. The 5' UTR is engineered to promote translation initiation—many therapeutic mRNAs have a Kozak sequence near the initiation codon. The 3' UTR is engineered to promote stability. The mRNA is capped in vitro—Cap1 and Cap2 are preferred—and it is polyadenlated (about 100 As).

The coding region resembles part of the coding region of a modified SARS-CoV-2 spike protein that binds to the ACE2 receptors. Production of this protein in human cells after injection elicits an antibody response that confers immmunity to the virus. The coding region may be modified to include specific codons that willl be preferentially translated in human cells using the modified nucleotides. The mRNAs in the current crop of vaccines are about 4300 bp long.

Lipid nanoparticles

An average of 1-10 mRNA molecules are enclosed in small lipd vesicles that protect the RNA from nucleases and facilitate entry into the cell by fusing with the plasma membrane. The composition of the Pfizer/BioNTech and Moderna lipid nanoparticles are known. The Pfizer/BioNTech vaccine uses nanoparticles developed by Acuitas, a Canadian company based in Vancouver, BC.1 Several other mRNA vaccines use the Acuitas technology. The Moderna vaccine uses the OnpattroTM delivery system developed for another purpose in 2019. As far as I can figure out, it incorporates features of the Acuitas system with modifications made at labs in Cambridge, MA (USA).

The problem with lipid nanoparticles is that you need a lipid composition that readily fuses with the cell membrane but is stable while circulatiing in the blood. It also has to be non-toxic. The collection of lipids in the particle are mostly ionizable complex lipids with multiple fatty acid-like chains that don't look anything like the typical triglycerides seen in the textbooks. One of the problems with these lipids is that the pKa of the ionizable groups have to be in the 6-7 range so that they aren't charged when circulating in the blood but can be readily protonated in the cell to facilitate the release of the mRNA. An example of one of the BioNTech lipids from Acuitas is shown below.

The lipid nanoparticles also contain ordinary cholesterol and a dispersive lipid called distearoylphosphatidylcholine (DSPC). The stability and solubility of the particles is enhanced by attaching polyethylene glycol chains to the outer surface and this is a possible cause for concern since some people are allergic to PEG. If you get one of these vaccinations you will be asked if you are allergic to PEG.

Here's a description of how the mRNA-containing lipid nanoparticles are assembled.

Figure 2. mRNA lipid nanoparticle assembly is achieved by (A) rapid mixing in a microfluidic or T-junction mixer of four lipids (ionizable lipid, DSPC, cholesterol, PEG–lipid) in ethanol with mRNA in an aqueous buffer near pH4. (B) When the ionizable lipid meets the aqueous phase, it becomes protonated at a pH ~5.5, which is intermediate between the pKa of the buffer and that of the ionizable lipid. (C) The ionizable lipid then electrostatically binds the anionic phosphate backbone of the mRNA while it experiences hydrophobicity in the aqueous phase, driving vesicle formation and mRNA encapsulation. (D) After initial vesicle formation, the pH is raised by dilution, dialysis or filtration, which results in the neutralization of the ionizable lipid, rendering it more hydrophobic and thereby driving vesicles to fuse and causing the further sequestration of the ionizable lipid with mRNA into the interior of the solid lipid nanoparticles. The PEG–lipid content stops the fusion process by providing the LNP with a hydrophilic exterior, determining its thermodynamically stable size, and the bilayer forming DSPC is present just underneath this PEG–lipid layer.

Developing the technology to produce effective lipid nanoparticles was a real challenge because not only did the appropriate strange lipids have to be manufactured but their ratio in the particle had to be carefully controlled. Then you had to figure out how to get the mRNA molecules inside and control the size of the particles. Then you had to complete clinical trials to show that the nanoparticles were safe. All of this research was completed before the beginning of the pandemic—it was not significantly helped by Operation Warp Speed or any of the similar programs supported by dozens of other countries. Those programs helped speed up the clinical trials and provided insurance to back up manufacturing the virus before the trials were complete. America's contribution was only a small part of this world-wide effort.

1. It's near the University of British Columbia and just a block from Tim Hortons!

Buschmann, M.D., Carrasco, M.J., Alishetty, S., Paige, M., Alameh, M.G. and Weissman, D. (2021) Nanomaterial delivery systems for mRNA vaccines. Vaccines 9:65-. [doi: 10.3390/vaccines9010065]

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