mRNA Vaccines (I)

choi

I write these two postings with laypersons in mind, but am unsure whether they understand the writing.


Modified mRNA Vaccines; Katalin Karikó, Drew Weissman. Lasker Foundation, Sept 24, 2021.
https://laskerfoundation.org/winners/modified-mrna-vaccines/

Quote:

"mRNA could, in principle, provide a means to turn a person's cells into factories for any desired protein. Such methodology might replenish essential substances that are in short supply or introduce microbial components as a vaccine. Furthermore, it offered numerous advantages over DNA-based strategies. Unlike DNA, mRNA would not threaten the recipient cell’s genomic integrity because it cannot integrate into the chromosome and interrupt resident genes or wreak other mutational havoc. [This quotation suggests that in lieu of one-off gene therapy (with correct DNA), patients with gene defects may received periodical intramuscular injections of mRNA. For example, hemophiliacs started receiving infusion of plasma in 1950s and of purified factors VIII or IX in 1970s. Insertion of a foreign DNA may cause cancers.]

"For decades, the tantalizing prospects of mRNA therapeutics remained theoretical due to practical impediments. Scientists did not develop ways to synthesize mRNA in a test tube until 1984. After that advance, they still needed to figure out how to deliver it to cells inside animals. Our bodies swarm with enzymes that chew up RNA, and even if the molecule survives the extracellular milieu long enough to encounter a target cell, it then encounters a formidable hurdle: the lipid bilayer. mRNAs are too large to diffuse across this membrane, and they also carry a negative charge [due to presence of phosphate ions in both RNA and DNA], which is rebuffed by the oily barrier. [All problems in this quotation is solved by using lipid nanoparticle to package mRNA (within the nanoparticle). See II in next posting.]  Although researchers made headway—in the late 1970s, they began to develop lipid-based materials that could package, protect, and transport mRNA to cells * * *

"by the early 1990s [starting 1990], she [Karikó] was a research assistant professor at the University of Pennsylvania * * * In 1997, immunologist Drew Weissman, a new faculty member at Penn [met her] * * * Natural encounters with foreign invaders spark transition of the dendritic cells [which together with macrophages are descendants of monocytes' dendritic cells are so named because their maturation causes branches to grow out of them, like a tree; Ancient Greek noun neuter déndron tree] into a mature form that propels T cells into action. * * * [in addition] mature dendritic cells spit out cytokines, signaling molecules that recruit and provoke other components of the body's defense system [which is bad for mRNA vaccine]. * * * As a first step, they wanted to discern characteristics of the mRNAs that rouse dendritic cells. Toward that end, the investigators tested RNAs from different sources. Bacterial RNA triggered a potent response; total mammalian RNA kindled a mild one [total RNA inside a human cell contains ribosomal RNA (80%), mRNA (5%) and transfer RNA]. When they homed in further, they found that RNA from human mitochondria, an organelle whose evolutionary origins trace to bacteria, also spurred dendritic cell activation. On the other end of the spectrum, mammalian transfer RNA (tRNA) stirred none. This surprising result provided the crucial clue to Karikó and Weissman's groundbreaking insight.  Four building blocks compose all RNAs: the nucleosides adenosine, uridine, guanosine, and cytidine. tRNAs are unusual in that they contain a high proportion of so-called modified nucleosides that differ in subtle ways from the standard ones. These variants carry extra chemical groups or have their components attached to one another through different atoms (Figure 1). Karikó and Weissman wondered whether modified nucleosides ]see illustration] would render the mRNAs immunologically inert. * * * [their experiments showed] the altered versions [of nucleosides in mRNA] failed to activate TLR7 and TLR8 [TLR stands for 'toll-like receptor'], and they published these game-changing data in 2005. Swapping uridine for its common relative, pseudouridine, was especially effective in preventing activation.

Note:
(1)
(a) Katalin Karikó
https://en.wikipedia.org/wiki/Katalin_Karikó
(1955- ; Hungarian; "grew up in Kisújszállás [a town whose population in 2002 was 13,000: en.wikipedia.org), Hungary. Her father was a butcher"/ PhD and postdoctoral research in Hungary; left for US in 1985)
(b) Drew Weissman
https://en.wikipedia.org/wiki/Drew_Weissman  (MD and PhD in 1987 from Boston University; "In 1997, Weissman moved to the University of Pennsylvania to start his laboratory")
(c) The 2005 paper:
Karikó K, Buckstein M and Weissman D, Suppression of RNA Recognition by Toll-Like Receptors: the Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity, 23: 165 (2005).
https://www.cell.com/immunity/fulltext/S1074-7613(05)00211-6

Quote:

Summary: "DNA and RNA stimulate the mammalian innate immune system through activation of Toll-like receptors (TLRs). DNA containing methylated CpG motifs, however, is not stimulatory.

Introduction: " * * * DNA containing unmethylated CpG motifs ['methylated cytidine in CpG motifs of DNA'], characteristic of bacterial and viral DNA, activate TLR9 (Hemmi et al., 2000). Double-stranded (ds)RNA, a frequent viral constituent, has been shown to activate TLR3 (Alexopoulou et al., 2001, Wang et al., 2004), single-stranded (ss)RNA activates mouse TLR7 (Diebold et al., 2004) [unless specified (mouse here), molecules referred to in this article were of human] * * * Ribosomal RNA, the major constituent (∼80%) of cellular RNA, contains significantly more nucleoside modifications when obtained from mammalian cells versus bacteria. Human rRNA, for example, has ten times more pseudouridine (Ψ) and 25 times more 2′-O-methylated nucleosides than bacterial rRNA, whereas rRNA from mitochondria, an organelle that is a remnant of eubacteria (Margulis and Chapman, 1998), has very few modifications (Bachellerie and Cavaille, 1998). Transfer RNA is the most heavily modified subgroup of RNA. In mammalian tRNAs, up to 25% of the nucleosides are modified, whereas there are significantly less modifications in prokaryotic tRNAs. Bacterial mRNA contains no nucleoside modifications, whereas mammalian mRNAs have modified nucleosides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine and many 2'-O-methylated nucleosides in addition to N7-methylguanosine (m7G), which is part of the 5′-terminal cap (Bokar and Rottman, 1998). The presence of modified nucleosides was also demonstrated in the internal regions of many viral RNAs including influenza, adeno, and herpes simplex; surprisingly, modified nucleosides were more frequent in viral than in cellular mRNAs (Bokar and Rottman, 1998) [whose significance is to be discussed in Discussion]. * * *

Discussion: "N6-methyladenosine (m6A) is the only base-modified nucleoside that is present in all RNA types, including rRNA, tRNA, and snRNA, as well as in mRNAs of cellular and viral origins. The methylation in m6A interferes with Watson-Crick base pairing, thus, its presence destabilizes RNA duplexes (Kierzek and Kierzek, 2003). This characteristic of m6A might explain why RNA containing m6A did not stimulate TLR3 (Figure 2B). m6A is present in mRNA of mammalian cells and RNA of viruses that replicate in the nucleus such as influenza, adenovirus, HSV, SV40, and RSV (Bokar and Rottman, 1998). In general, m6A modifications were found internally, mostly in coding sequences, and viral mRNA usually contained significantly more m6A than cellular mRNA (Bokar and Rottman, 1998). Interestingly, Rous sarcoma virus replicated similarly with and without m6A when tested in cell culture (Kane and Beemon, 1987), therefore no function could be assigned to m6A in this viral mRNA. It is tempting to speculate that the presence of m6A in viral RNA might serve the virus by allowing it to avoid immune activation. This suggestion is strengthened by considering that the frequency of m6A modifications found in viral mRNAs, up to eight per a 1.8 kb-long segment of influenza RNA (Narayan et al., 1987), is sufficient to suppress the capacity of RNA to activate DCs (Figure 5). Because those early studies with viruses were performed in cell culture and not in animals, the immune suppressive effect of m6A might have been missed.

(i) CpG site
https://en.wikipedia.org/wiki/CpG_site  
("Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosines)

5-methylcytosines has only ONE methyl group, so you may guess that the carbon in the ring the methyl group is attached to is 5. but here is the numbering system in a pyrimidine.
https://en.wikipedia.org/wiki/Pyrimidine
(upper right corner of the graphic)
, where oistion 1 is to connect with ribose.
(ii)
(A) N6-methyladenosine
https://en.wikipedia.org/wiki/N6-Methyladenosine
(B) purine
https://en.wikipedia.org/wiki/Purine

Note the numbering system in the pyrimidine. However, you will notice that the N6 in N6-methyladenosine is not part of the pyrimidine ring. So, why N6?  In other words, where does the name N6 come from?  I am a biologist by training, but have not heard of N6. It took me about half an hour to know what it means. See
Huang JL, Chen Zym Chen X, Chenb H, Cheng ZX and Wang ZX, The role of RNA N6-Methyladenosine Methyltransferase in Cancers. Molecular Therapy Nucleic Acids, 23: 887 (Mar 5, 2021; review)
https://www.cell.com/molecular-t ... fulltext/S2162-2531(20)30402-9
("m6A in RNA is an epigenetic modification in which a hydrogen atom (–H) connected to the sixth nitrogen atom (N6) on adenine is replaced by a methyl group (–CH3) (Figure S1)")


(2) The Story of Toll. It started with Drosophila (fruit fly) and ends in human.
(a) Elizabeth R Gavis, Gurken Meets Torpedo for the First Time. Current Biology, 5: 1252 (1995; under the heading "Pattern formation")
https://reader.elsevier.com/reader/sd/pii/S0960982295002508
("The development of embryonic polarity in the fruitfly Drosophila melanogaster requires the formation of gradients of determinant molecules [(Reference No) 1]. Opposing gradients of Bicoid (Bcd) and Nanos (Nos) proteins emanate from the embryonic poles and determine polarity along the anterior-posterior axis * * * The Drosophila oocyte develops within an egg chamber consisting of an oocyte and its 15 sister nurse cells, which derive from the divisions of a single germline stem cell, and a surrounding epithelium of somatic follicle cells [4]. Anterior-posterior asymmetry becomes evident before dorsal-ventral asymmetry, with the movement of the oocyte to one end of the egg chamber (the future posterior end) early in oogenesis. * * * These intercellular communication events were uncovered by analyses of mutations in several genes that affect both follicle-cell identity and oocyte polarity These are 'maternal-effect' mutations, the phenotypes of which are manifested in oocytes produced by homozygous female [read: mother] flies")
(i) English dictionaries spell fruit fly, rather than fruitfly. I will use the former. Not forewing born, Gavis is based in Princeton University.
(ii) In fruit fly, an egg (outside the mother) knows anterior-posterior or dorsal-ventral axis due to a gradient (highest to lowest concentration) of (different sets of) proteins in each axis. For the former, view Figures 3 (caption: "mRNA distributions") and 4 (caption: :protein distributions") in Drosophila embryogenesis.
https://en.wikipedia.org/wiki/Drosophila_embryogenesis

This Wiki page deals with an egg outside of fruit fly mother already. The next -- (iii) -- concerns an egg developing inside the mother's ovary (and hence at an earlier stage; without eggshell yet).
(iii) "an oocyte and its 15 sister nurse cells, which derive from the divisions of a single germline stem cell"

In fruit fly, one germline stem cell divides four times to produce 16 cells (1, 2, 4, 8, 16), out of which one is oocyte and the other 15 nurse cells. See egg cell
https://en.wikipedia.org/wiki/Egg_cell

(section 4 Other organisms: "Drosophila oocytes develop in individual egg chambers that are supported by nurse cells and surrounded by somatic follicle cells. The nurse cells are large polyploid cells that synthesize and transfer RNA, proteins, and organelles to the oocytes. This transfer is followed by the programmed cell death (apoptosis) of the nurse cells. During oogenesis [as opposed to embryogenesis in (ii) above], 15 nurse cells die for every oocyte that is produced")

In human, two meioses generate an egg and three polar bodies that soon die.
(b) Hereafter, I will ignore anterior-posterior axis and concentrate on emergence of dorsal-ventral axis in a Drosophila oocyte.
(i) Firstly, names of two Drosophila genes: toll and .
(A) Sarah Zhang, Consider the Fruit Fly; Modern genetics would not be possible without the humble fruit fly. The Atlantic, February 2018
https://www.theatlantic.com/scie ... -drosophila/553967/
(This is a book review on Stephanie Elizabeth Mohr First in Fly; Drosophila research and biological discovery. Harvard Univ Press, 2018: "In college, I worked briefly in a fruit-fly lab, where I spent most of my time just keeping different fly strains alive. It was not difficult—as anyone with a fruit-fly infestation can tell you * * * In the 1970s, Christiane Nüsslein-Volhard and Eric Wieschaus in [European Molecular Biology Laboratory (EMBL) at] Heidelberg, Germany, were studying a * * * patterns [in oo- and embryo-genesis] of fruit-fly larvae. They performed what is called a 'forward genetic screen'—in which tens of thousands of male [and vice versa (especially for maternal-effect mutations)] fruit flies are fed a chemical that induces mutations and then individually mated with a female. Nüsslein-Volhard and Wieschaus then spent a year sitting side by side at the microscope, looking for individual mutants * * * Some of the most memorable ones [gene names] in Mohr's book include: * * * spätzle: Discovered in fruit flies whose larvae are irregularly shaped like the German noodle. In fruit flies, spätzle makes a molecule that binds to Toll proteins, named after Christiane Nüsslein-Volhard's expression 'Das ist ja toll!' ('That's amazing' in German")
• Professor Christiane Nüsslein-Volhard. "What Is Biotechnology?" undated
https://www.whatisbiotechnology. ... ry/Nusslein-Volhard
("Christiane Nüsslein-Volhard is the daughter of Rolf Volhard, an architect, and Brigitte Haas Volhard, a nursery school teacher. * * * Nüsslein-Volhard married young, in the mid-1960s [she was born in 1942]. She met her husband [not named] while studying at the Johann-Wolfgang-Goethe University in Frankfurt. The couple did not remain married for long and did not have any children. Despite the brevity of her marriage, Nüsslein-Volhard retained her new hyphenated name. This was because she had already begun to publish under that name")
• spätzle
https://en.wikipedia.org/wiki/Spätzle
(section 1 Etymology)

Recall that a German noun always has its first letter capitalized.

• German-English dictionary:
* das:
(article): "the" (see table)
(pronoun): "this, that, it"
https://en.wiktionary.org/wiki/das
* For ist, see
sein (irregular verb: third-person singular present ist): "to be"
https://en.wiktionary.org/wiki/sein
* ja (adv): "1: yes; 2: (intensifier) obviously; certainly; of course; really"
https://en.wiktionary.org/wiki/ja

Remember that the letter j in German is always pronounced like y (as in yes) in English.
* toll (adj; cognate with English dull): "(colloquial) great, nice, wonderful"
https://en.wiktionary.org/wiki/toll

Take notice that (Modern) English noun toll and adjective dull are derived both from Old English: tol/toll and dol, respectively.
(B) Juliane, Spaetzle. Boston University Medical Center (BUMC) Postdics Blog, Oct 9, 2013
https://blogs.bu.edu/bumcpostdocs/2013/10/09/spaetzle/
("This is a plate of spätzle [photo 1].  It’s the German version of macaroni and cheese, which has to be made from scratch every single time. Here I zoomed in onto a single one [photo 2]. And underneath the close up is a picture of a Drosophila embryo missing the gene spätzle [photo 4]. Can you see the similarity?")
• How to Make Homemade Spaetzle. Super Mom No Cape, Oct 4, 2011
https://www.supermomnocape.com/how-to-make-homemade-spaetzle/
("To make Spaetzle using a Spaetzle maker [sectional heading]: Spoon the batter into the hopper of the spaetzle maker.  Move the hopper back and forth across the grater. * * * To make spaetzle the traditional way (without a spaetzle maker): * * * Holding the board over the boiling water, use a knife to cut off bits of the batter and drop them into the water")
• How to Make Spaetzle. Uploaded by cargar1218 on Oct 9, 2010
https://www.youtube.com/watch?v=pW1TJWei6Uo

At 2:30 (2 minutes 30 seconds) into the video, a hopper was assembled onto the grater.
(c) Trudi Schüpbach (born in Germany but at the time of this writing was at Princeton Univ), Genetic Screens to Analyze Pattern Formation of Egg and Embryo in Drosophila: A Personal History. Annual Review of Genetics, 53: 1 (2019)
https://www.annualreviews.org/do ... genet-112618-043708

Introduction: [paragraph 1:] For Drosophila geneticists, one of the enjoyable tasks in large-scale mutagenesis experiments is thinking of names that imaginatively describe each new mutant phenotype. At the beginning of my career as an independent scientist, I conducted a large-scale mutagenesis screen to isolate female sterile and maternal-effect mutations on the second chromosome of Drosophila. The screen was successful and led to the identification of many loci with striking phenotypes affecting oogenesis and early embryonic development. What name would best describe maternal-effect mutations that eliminate the germplasm in the developing egg such that the resulting progeny are sterile and the original mutant female therefore remains grandchildless? We thought of royal families that died out because of lack of offspring—Tudor was the first that came to mind; Vasa, Staufen, and Valois followed soon after. How to name mutations that cause the eggs laid by the female to be elongated and more or less uniformly patterned in the circumferential dimension? gurken, which means 'cucumber' in German, was soon followed by zucchini, aubergine, okra, squash, and later, cornichon [FRENCH noun masculine for cucumber], as devised by Siegfried Roth. Finding a descriptive name for mutants and thus for the corresponding genes confers a certain emotional attachment to those genes, as probably many geneticists working with flies or zebrafish or mice would agree.

Figure 1 [caption:] Trudi Schüpbach and Eric Wieschaus at a conference in Greece in 1980. Photo provided by Trudi Schüpbach.

"Regulation of the Signal in the Germline [sectional heading] [paragraph 1:] One of the exciting aspects of science is that solving one question almost always leads to many more questions, often venturing into fields that one might not have initially chosen for study. When F. Shira Neuman-Silberberg cloned gurken, she found that the gurken messenger RNA (mRNA) was localized to one corner of the developing oocyte, in close proximity to the oocyte nucleus (32). In mid[-]oogenesis, the oocyte nucleus moves from an initially central position at the posterior of the
small egg chamber to an asymmetric cortical position at the anterior. The site where the nucleus anchors determines the future dorsal side of the egg and embryo. Gurken protein adjacent to the nucleus signals to the overlying follicle cells and activates the Egfr in a restricted set of follicle cells (Figure 2). The downstream ERK [which stands for extracellular signal-regulated kinases, which you need not know] pathway then activates several genes in these follicle cells that pattern the dorsal eggshell while repressing expression of the gene pipe (52). David Stein demonstrated in elegant experiments how this asymmetrical expression of pipe on the ventral side initiates a new signal that sets up the dorsal-ventral axis of the embryo via processing of Spätzle and activation of Toll (53, 54). * * *

Please view Figure 2; maybe you may guess its meaning.
(d) What did a loss=of-function mutation of gurgen look like?

Wasserman J and Freeman M, An Autoregulatory Cascade of EGF Receptor Signaling Patterns the Drosophila Egg. Cell, 95: 355 (1998)
web.mit.edu/be.400/www/Papers/Wasserman_Cell_1998.pdf
(Figure 1. Spitz Is Required in the Follicle Cells[:] (A ['Wild type' -- which means normal] and B) The dorsal surface of the anterior of the Drosophila egg [outside the mother] has two respiratory appendages that emerge from either side of dorsal midline * * * (G) Complete loss of EGFR function in a gurken mutant (grkHK [Trudi Schüpbach never explained in her publications why she named gurken mutant this way or that way]) causes loss of all dorsal appendage")
(e) Christiane Nüsslein-Volhard (age 53), Eric Wieschaus (age 48; American) and Edward B Lewis (age 73; American; also studied fruit fly] were awarded with Nobel Prize in Physiology or Medicine in 1995.


(3)
(a) Nüsslein-Volhard and a postdoc from her lab, Kathryn V Anderson (American), cloned the toll gene in 1985 (from fruit fly). More than a decade would pass before toll was found to be involved in innate immunity of fruit fly. For the latter, see
Lemaitre B, Nicolas E, Michaut L, Reichhart J-M and Hoffman JA, The Dorsoventral Regulatory Gene Cassette spätzle/Toll/cactus Controls the Potent Antifungal Response in Drosophila Adults. Cell, 86: 973 (1996).
https://www.cell.com/cell/fulltext/S0092-8674(00)80172-5
(b) Salminen TS and Vale PF, Drosophila as a Model System to Investigate the Effects of Mitochondrial Variation on Innate Immunity. Frontiers in Immunology, 11: 521 (2020)
https://www.frontiersin.org/arti ... mmu.2020.00521/full
("Drosophila does not possess acquired/adaptive immunity [namely, fruit fly does not produce antibodies] and it relies on humoral and cell-mediated innate immunity for its defense against pathogens, such as bacteria, viruses, fungi, and parasites. * * * In Drosophila, the humoral innate immune response to bacterial pathogens is characterized by the production and release of a cocktail of AMPs [antimicrobial peptides] into the hemolymph. This response is driven by two evolutionarily conserved and largely independent pathways, Immune deficiency (IMD) and Toll pathways (116). The Toll pathway is induced by bacteria containing LYS-type peptidoglycan in their cell walls (mainly Gram-positive bacteria), while the IMD pathway is induced by DAP-type peptidoglycan (mainly Gram-negative) bacteria. These pathways culminate in the translocation of NF-κB dimers [you need not know what NF-κB is, except to know that both lead to the common pathway] to the nucleus leading to infection-specific upregulation of AMPs targeted to clear the infection (117–119)")
(c) Human toll-like receptors (TLRs) do not have a ligand corresponding to fruit fly's spätzle; rather they (human TLRs) "are activated directly by bacterial, fungal, or viral components." (I quote a scientific article.)

In fruit fly, both development (in an egg) and innate immunity (excluding IMD pathway) involves the same spätzle-toll pathway. See
Valanne S et al, The Drosophila Toll Signaling Pathway. Journal of Immunology, 186: 649 (2011)
https://www.jimmunol.org/content/186/2/649.long
, whose Figure 1 had a caption that said in full:

"Extracellular cleavage of Spz leading to Toll pathway activation. In early embryogenesis, the protease cascade Gastrulation Defective-Snake activates the protease Easter, which cleaves full-length Spz. In the immune response, three protease cascades lead to the activation of SPE to cleave full-length Spz; the Persephone (PSH) cascade senses virulence factors and is activated by live Gram-positive bacteria and fungi. The other two cascades are activated by pattern recognition receptors binding cell wall components from Gram-positive bacteria and fungi, respectively. All cascades converge at ModSP-Grass for downstream activation of SPE. Upon proteolytical processing, the Spz prodomain is cleaved, exposing the C-terminal Spz parts critical for binding of Toll. Spz binding to the Toll receptor initiates intracellular signaling."

Spz = spätzle
A series oor cascade of serine proteases activate Spz by clipping off a part of its predecessor. These serine protease are: Gastrulation Defective, Snake, Easter, ModSP (for Modular serine protease), grass, Sphinx/2, Spirit, Spheroide, Persephone (PSH), and Pipe.
SPE = spätzle-processing enzyme