mRNA Vaccines (II)

choi

Lynda M Stuart, In Gratitude for mRNA Vaccines. New England Journal of Medicine, _: _ (online publication Sept 24, 2021).
https://www.nejm.org/doi/full/10.1056/NEJMcibr2111445

Quote:

"Before 2020, the average time from conception of a vaccine to licensure was 10 to 15 years; the shortest time (for the mumps vaccine) was 4 years. The development of a vaccine for coronavirus disease 2019 (Covid-19) in 11 months was therefore an extraordinary feat and was made possible by years of basic research on new vaccine platforms, most notably messenger RNA (mRNA).

"real-world application of DNA vaccination has been limited, initially because of safety concerns regarding DNA integration [into host's DNA] and later because of the poor scalability of efficient delivery of the DNA into the nucleus. In contrast, despite being prone to hydrolysis [which prevents infusion into blood stream, where enzymes (nucleases) immediately chop them off], mRNA appeared to be more tractable because the nucleic acid did not need to be delivered into the nucleus; it is functional in the cytosol. However, decades of basic research performed by Weissman and Karikó, initially in their own laboratories and then after licensing to two biotechnology companies (Moderna and BioNTech), were needed for the realization of mRNA vaccines.  They had to overcome several hurdles. mRNA is recognized by innate immune-system pattern-recognition receptors (Figure 1), including the toll-like receptor family members (TLR3 and TLR7/8, which sense double-stranded RNA and single-stranded RNA, respectively) and the retinoic acid–inducible gene I protein (RIG-I) pathway, to induce an inflammatory response and cell death. (RIG-I is a cytosolic pattern-recognition receptor that recognizes short double-stranded RNA and activates type I interferon and thus the adaptive immune system.) Consequently, injection of mRNA in animals led to shock [I fail to find anything to this effect in medical literature]

"The final breakthrough was the determination of how best to package the mRNA to protect it from hydrolysis and to deliver it to the cytosol of the cell. Various mRNA formulations had been tested in a number of vaccines against other viruses. In 2017, such testing led to clinical evidence of an mRNA vaccine that, when encapsulated and delivered by a lipid nanoparticle, boosted immunogenicity while retaining a manageable safety profile.8 Supporting studies in animals showed that lipid nanoparticles targeted antigen-presenting cells in the draining lymph node and also adjuvanted the response by inducing the activation of a particular type of follicular CD4 helper T cell.9

Note: This posting will concentrate on lipid nanoparticle (LNP).
(1) Liposomes and Lipid Nanoparticles as Delivery Vehicles for Personalized Medicine. Indianapolis: Exelead (which custom-makes LNP for pharmaceutical clients), undated
https://www.exeleadbiopharma.com ... rsonalized-medicine
("Some [probably all] LNPs assume a micelle-like structure, encapsulating drug molecules in a non-aqueous core [hence LNPs are also called solid LNPs, for unlike liposomes, LNPs do not contain an aqueous core]")
(a) Do not read (because the page is not informative). View illustrations only to distinguish liposome and micelle.
(n; First Known Use 1881; from New Latin micella, from Latin [noun feminine] mica [crumb])
https://www.merriam-webster.com/dictionary/micelle
(pronunciation)

You need not know its definition, as a picture is worth a thousand words.  

So, a liposome is made up of lipid bilayer (the same as cell membrane), with aqueous solution inside. On the contrary, a micelle (and LNP also) has one layer (not two) surrounding the particle, whose content (not much in a micelle but more in LNP) is ""non-aqueous" or oily (lipid soluble).
(b) A molecule of triglyceride is depicted schematically as a ball with three tentacles. A triglyceride is composed of one glycerol (with three -OH groups) and three fatty acids (which may or may not be the same fatty acids, each having one -COOH group), via ester bonds. The long aliphatic chain in in each fatty acid is hydrophobic (oilly), whereas the glycerol backbone is hydrophilic. In other words, a triglyceride is polar: one end hydrophilic and the other end hydrophobic.  

(2) Ryan Cross, Without These Lipid Shells, There Would Be No mRNA Vaccines for COVID-19; Fragile mRNA molecules used in COVID-19 vaccines can't get into cells on their own. They owe their success to lipid nanoparticles that took decades to refine. c&en (published by American Chemical Society), volume 99 (Mar 6, 2021),
https://cen.acs.org/pharmaceutic ... mRNA-vaccines/99/i8

Quote:

"LNPs used in the COVID-19 vaccines contain just four ingredients: ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA, pegylated [also written PEGylated] lipids that help stabilize the particle, and phospholipids and cholesterol molecules that contribute to the particle's structure. Thousands of these four components encapsulate mRNA, shield it from destructive enzymes, and shuttle it into cells, where the mRNA is unloaded and used to make proteins.

"Over more than 3 decades, promising lipids [including liposome; Ancient Greek noun neuter sôma body] studied in the lab often failed to live up to their potential when tested in animals or humans.

"the first drug based on an LNP was approved by the US Food and Drug Administration for a rare genetic disease in 2018 [Alnylam Pharmaceuticals' Onpattro to treat hereditary transthyretin-mediated amyloidosis, with small interfering RNA (siRNA; 'small' because these synthetic RNAs are short (20-27 base pairs of double-stranded RNA) based on RNA interference (RNAi); the company is based in Cambridge, Mass and 'named after Alnilam [sic], a star in Orion's belt': en.wikipedia.org]

"Modern LNPs can be traced back to work on simpler systems called liposomes, hollow lipid spheres often made of just two or three kinds of lipids.

"At the time, scientists were enamored by advances in genetics that were promising to cure diseases by giving someone new genes or turning disease-causing genes off [with siRNA]. Figuring out how to deliver these nucleic acid therapies—either DNA or RNA—into cells was a major challenge and required something more sophisticated than a conventional liposome. Cullis knew that adding positively charged lipids to the liposomes would help balance the negatively charged nucleic acids, but there was a problem. 'There are no cationic lipids in nature,' [University of British Columbia nanoparticle scientist Pieter] Cullis says. 'And we knew we couldn't use [man-made] permanently positively charged lipids because they are so damn toxic.' Those lipids would rip cell membranes apart, he adds.  A solution came from new lipids that were charged only under certain conditions. During the late '90s and through the first decade of the 2000s, Cullis, his colleagues at Inex Pharmaceuticals, and the Inex spin-off Protiva Biotherapeutics [based in City of Burnaby (eastern neighbor of Vancouver), British Columbia] developed ionizable lipids that are positively charged at an acidic pH [as in endosome; to be explained 2 quotations down] but neutral in the blood. The group also created a new way to manufacture nanoparticles with these lipids, using microfluidics to mix lipids dissolved in ethanol with nucleic acids dissolved in an acidic buffer. When the streams of those two solutions merged, the components spontaneously formed lipid nanoparticles, which, unlike the hollow liposomes, were densely packed [inside] with lipids and nucleic acids.

"Pegylated lipids, in which polyethylene glycol (PEG) strands are attached to lipid heads, have several functions in a nanoparticle. PEG helps control the particle size during formulation, prevents the particles from aggregating in storage, and initially shields the particles from being detected by immune system proteins in the body, according to James Heyes, a former Protiva scientist. * * * But PEG also has liabilities. * * *

"LNPs take advantage of a natural process called receptor-mediated endocytosis [see LDL in the latter part of this quotation] to get into cells, Madden explains. Upon binding to a cell, the nanoparticle becomes encapsulated in an even bigger lipid bubble—an organelle called an endosome. The endosome's acidic interior protonates the heads of the ionizable lipids, making them positively charged. That positive charge triggers a change in the shape of the nanoparticle, which scientists think helps it break free from the endosome and ultimately release its RNA cargo into the cell's cytoplasm. Once released, the RNA is free to do its job.  The most effective nanoparticles were ones that the body mistook as low-density lipoprotein (LDL) cholesterol—commonly called bad cholesterol. Proteins [soluble protein in the blood, exemplified by apolipoprotein E (apoE)] that recognize LDL cholesterol in the blood bound to some of Alnylam's nanoparticles and carried them to LDL receptors on liver cells, which then caused the cells to engulf the nanoparticles in an endosome. It was the kind of complex interplay that studies in a petri dish missed.

"By 2010, Alnylam had landed on a winning ionizable lipid known as MC3. * * * Alnylam used the new formulation in patisiran (Onpattro), its treatment for a rare disease called hereditary transthyretin-mediated amyloidosis. In 2018, patisiran became the first approved siRNA drug and the first approved therapy delivered via LNPs.

"The off-the-shelf LNP formulations designed for siRNA worked for mRNA occasionally but not very well, says Romesh Subramanian, who led a team at Alexion Pharmaceuticals that worked on mRNA therapies with Moderna from 2014 to 2017. siRNA molecules are like short rods, with two rows of about 20 nucleotides each, he explains. mRNA, in contrast, can easily span thousands of nucleotides, wind into complex shapes, and change the properties of the LNP in ways that are hard to predict.  After realizing that MC3 wouldn't cut it for mRNA delivery, Moderna invested significant resources into building a better ionizable lipid. * * * For its COVID-19 vaccine, Moderna ultimately used an [proprietary] ionizable lipid that it calls SM-102, which it first described in a 2018 study on alternatives to MC3. Pfizer and BioNTech licensed an ionizable lipid called ALC-0315 from Acuitas [Therapeutics at Vancouver, BC]. * * * Those [three aforesaid] ionizable lipids, which are remarkably similar in structure, were discovered while the firms were optimizing LNPs for systemic administration [ie, intravenous or infusion] and delivery to the liver—not the intramuscular injection of a vaccine. Experts point out that optimizing [from fit for iv (as is now, for there is no better ionizable lipid) to im (intramuscular)] the nanoparticles for vaccination could lead to shots that require lower doses [than what is used currently in im injections], which could ease the manufacturing burden amid a pandemic.

"Right now, intravenous injections of nanoparticles can easily reach the liver, and intramuscular injections for vaccines are taken up by immune cells [in the lymph nodes, where dendritic cells take up LNPs and hence mRNA inside].

Note:
(a) polyethylene glycol
https://en.wikipedia.org/wiki/Polyethylene_glycol
(chemical formula)
(b)
(i) "Upon binding to a cell, the nanoparticle becomes encapsulated in an even bigger lipid bubble—an organelle called an endosome. * * * Proteins [soluble protein in the blood, exemplified by Apolipoprotein E] that recognize LDL cholesterol in the blood bound to some of Alnylam's nanoparticles and carried them to LDL receptors on liver cells, which then caused the cells to engulf the nanoparticles in an endosome."

When a human ingests (eats) lipids, the lipids were absorbed in intestine, carried in the blood as LDL by apoE and delivered to liver, whose cell membrane contains LDL receptors. Once LDL is docked to LDL receptor, both are internalized into the cell and go directly to endosome, to be braoken apart there for ingredients. Certain LNPs somehow are treated similarly and delivered to liver endosome.   
(ii) “Certain LNPs”?
(A) Reference No 9 in Lynda M Stuart's NEJM essay is
Pardi N et al, Nucleoside-Modified mRNA Vaccines Induce Potent T Follicular Helper and Germinal Center B cell Responses. Journal of Experimental Medicine, 215: 1571 (2018).
https://rupress.org/jem/article/ ... nes-induce-potent-T
, whose Figure 1 showed images from "IVIS Spectrum imaging system (Caliper Life Sciences)." IVIS is the trademark of this machinery, which did in vivo work (that is, the mice were not killed before the imaging). The images clearly showed lymph nodes, not liver, were successfully targeted.
(B) Zukancic D et al, The Importance of Poly(ethylene glycol) and Lipid Structure in Targeted Gene Delivery to Lymph Nodes by Lipid Nanoparticle. Pharmaceutics, 12: 1068 (Nov 9, 2020)
https://www.mdpi.com/1999-4923/12/11/1068/htm
("Targeted delivery of nucleic acids to lymph nodes is critical for the development of effective vaccines and immunotherapies. However, it remains challenging to achieve selective lymph node delivery. Current gene delivery systems target mainly to the liver and typically exhibit off-target transfection at various tissues. Here we report novel lipid nanoparticles (LNPs) that can deliver plasmid DNA (pDNA) to a draining lymph node, thereby significantly enhancing transfection at this target organ, and substantially reducing gene expression at the intramuscular injection site (muscle). * * * However, current LNP technology delivers nucleic acids to only a few tissues such as liver and muscle, limiting their potential applications and the effectiveness of nucleic acid vaccines or cancer immunotherapies [18,19]")

transfection
https://en.wikipedia.org/wiki/Transfection  
("deliberately introducing naked or purified nucleic acids into eukaryotic cells. * * * The word transfection is a portmanteau of trans- and infection")
(c) "The endosome's acidic interior protonates the heads of the ionizable lipids, making them positively charged"

Triglycerides are neutral, carrying neither negative or positive charges. But the three man-made ionizable lipid (see the second figure in the c&en article) has each a tertiary nitrogen atom (tertiary means three bonds), which may attach an hydrogen ion in an acidic environment (similar to a molecule of ammonia to become an ammonium ion).
(d) The first figure of this c&en article shows a cut-out of an LNP. LNP's boundary consists of one layer of lipid, not two; its content is tons of smaller particles with mRNAs inside. But the ORIENTATION of the lipid in LNP boundary and boundary of smaller particle are opposite, because water and oil do not mix, rendering the hydrophobic tails of lipid next to one another spontaneously.