AMB 14- RNA Therapeutics

Comprehensive vocabulary flashcards detailing concepts, molecular mechanisms, formulations, and regulatory advances in RNA therapeutics and vaccines.

Lipid nanoparticles (LNPs)

Spherical delivery vehicles with a diameter of approximately $50\,\text{nm}$ composed of ionizable lipids, phospholipids, cholesterol, and PEGylated lipids, engineered to protect nucleic acids from RNase degradation and enable intracellular delivery.

Pseudouridine ($\Psi$)

A modified nucleoside isomer of uridine synthesized via pseudouridine synthase that suppresses Toll-like receptor recognition and diminishes PKR activation, thereby improving mRNA stability and translational output.

1-methylpseudouridine ($\text{m1}\Psi$)

A chemically modified nucleoside used as a $100\%$ replacement for uridine in the BNT162b2 and mRNA-1273 COVID-19 vaccines to decrease immunogenicity and enhance translation even more effectively than pseudouridine.

Protein Kinase R (PKR)

An intracellular sensor kinase that dimerizes and autophosphorylates upon detecting unmodified foreign RNA, subsequently phosphorylating eIF2$\alpha$ to inhibit protein translation as part of the integrated stress response.

eIF2$\alpha$

A eukaryotic translation initiation factor whose phosphorylation by activated PKR arrests protein translation during the cellular antiviral defense response.

ALC-0315

The synthetic ionizable lipid constituent of the lipid nanoparticle delivery system utilized in the BioNTech/Pfizer BNT162b2 mRNA vaccine.

SM-102

The proprietary ionizable lipid constituent employed in the lipid nanoparticle formulation of the Moderna mRNA-1273 vaccine.

ALC-0159

The PEGylated stealth lipid component incorporated in the BioNTech/Pfizer BNT162b2 lipid nanoparticle to provide immune cloaking and maintain nanoparticle stability.

PEG2000-DMG

The PEGylated stealth lipid used in the Moderna mRNA-1273 lipid nanoparticle formulation to enhance in vivo half-life and colloidal stability.

Distearoylphosphatidylcholine (DSPC)

A structural helper phospholipid included alongside cholesterol in lipid nanoparticles to form and stabilize the lipid bilayer shell.

K986P and V987P substitutions

Two stabilizing proline substitutions introduced into the SARS-CoV-2 full-length Spike glycoprotein encoded in both the BNT162b2 and mRNA-1273 vaccines to maintain the prefusion conformation.

Endosomal escape

The process by which internalized lipid nanoparticle-encapsulated mRNA escapes endosomal acidification and disruption into the host cytoplasm to be translated.

Self-amplifying RNA (saRNA)

An engineered RNA construct containing alphavirus nonstructural genes ($\text{nsP1-4}$) encoding an RNA-dependent RNA polymerase (RdRP) complex that directs intracellular replication of the vaccine immunogen.

Circular RNA (circRNA)

A closed, covalently linked single-stranded RNA loop lacking free $5^\prime$ and $3^\prime$ ends, providing exonuclease resistance, functioning as a sponge for miRNAs or RNA-binding proteins, and translating via cap-independent initiation.

Back-splicing

A non-canonical splicing process in which a downstream $5^\prime$ splice donor site joins to an upstream $3^\prime$ splice acceptor site to generate circular RNA containing a back-splice junction (BSJ).

Internal Ribosome Entry Site (IRES)

A specialized RNA structure that recruits the 40S ribosomal subunit to facilitate cap-independent initiation of translation in circular RNAs or viral transcripts.

Poly(A) tail mimetics

Synthetic oligonucleotide therapeutics consisting of an antisense guide linked to a poly(A) tail mimetic, designed to bind an mRNA $3^\prime$ UTR and prevent deadenylation in haploinsufficiency disorders.

Onpattro (patisiran)

The first FDA-approved RNAi (siRNA) lipid nanoparticle formulation, approved for the treatment of polyneuropathy caused by hereditary transthyretin-mediated amyloidosis in adults.

Duxil

The first lipid nanoparticle-encapsulated pharmaceutical formulation approved by the FDA in 1995.

KC2 and MC3

Ionizable cationic lipids discovered in 2010 and 2012, respectively, that advanced the efficacy and safety profile of lipid nanoparticles for nucleic acid delivery.

Katalin Karikó

Biochemist whose seminal work revealed that incorporating modified nucleosides into synthetic mRNA suppresses Toll-like receptor recognition and avoids PKR-mediated translational inhibition.

Kizzmekia S. Corbett

Viral immunologist whose structural design of coronavirus Spike protein immunogens and prototype pathogen research enabled the rapid generation of COVID-19 mRNA vaccines.

AMT-130

A clinical gene therapy developed by uniQure that delivers microRNA designed to silence the expression of the mutated gene that drives Huntington's disease.

Cap 1 structure

A mature eukaryotic $5^\prime$ mRNA cap feature that prevents innate immune detection; incorporated into BNT162b2 via a co-transcriptional Cap 1 analogue or generated enzymatically in mRNA-1273.

Stealth lipids

PEGylated lipids situated on the outer surface of lipid nanoparticles that provide immune evasion, prevent premature clearance, and prolong circulation half-life in vivo.

ILO 1- You understand the hurdles that limited RNA therapeutics

•RNA stability (it is very unstable and modifications (like swapping regular uridine for the pseudouridine makes it more stable and resistant to immediate destruction by the body's defenses.

•Delivery

•Immunogenicity (However, when scientists use synthetic modifications (like swapping regular uridine for the pseudouridine we discussed earlier), it becomes significantly more stable and resistant to immediate destruction by the body's defenses.)

ILO 2- You know what changes enabled new mRNA vaccines

•Lipid nanoparticles (LNPs)- microscopic bubbles of fat that act as protective delivery vehicles to transport genetic material (like mRNA) safely into your cells.

•Immunogenicity solved! Ψ

Ψ Pseudouridine (abbreviated by the Greek letter psi- Ψ) - an isomer of the nucleoside uridine in which the uracil is attached via a carbon-carbon instead of a nitrogen-carbon glycosidic bond.

Occurs naturally in our RNAs, put on by enzymes. Still pairs with A, but can influence RNA folding. Present in rRNA and tRNAs, snRNA snoRNAs.

ILO 3- You can apply your knowledge of RNA regulation to understand the features and design of mRNA vaccine platforms

mRNA vaccines more frequently used because of the urgency- Covid-19

Two Teams:

•BioNtech (inc. Kariko) + Pfizer

•NIH + Moderna

Both used

•1-methylpseudouridine (m1Ψ) - lowers unwanted immunogencity even more than Ψ

•Spike sequence (1,273-amino-acid homotrimeric structural protein encoded by the viral genome) codon-optimized for improved expression

ILO 4- You can provide examples of assays to test RNA therapeutic efficacy

• RT-qPCR / ddPCR: Measures mRNA knockdown or upregulation.

• Western Blot / ELISA: Quantifies target protein expression levels.

• Dual-Luciferase Assay: Verifies target sequence binding and silencing efficiency.

• In Vivo Imaging (IVIS): Tracks therapeutic translation and distribution in live models.

• CellTiter-Glo / Flow Cytometry: Measures downstream cellular survival and functional rescue.

ILO 5- You can describe other types of RNA medicines

We can also use RNA medicines to:

•Treat cancers – encode tumour-associated antigens (TAAs)/neoantigens

•Treat autoimmune diseases – retrain immune response to autoantigen

•Rare disease gene replacement (eg CF)

•Reverse tissue damage (eg VEGF)

Beyond vaccines, modified mRNA can be engineered to express specific clotting factors in liver cells to treat genetic bleeding disorders like hemophilia, which requires long-term expression strategies rather than the brief immune response needed for a vaccine.

Uridine is converted to pseudouridine by pseudouridine synthase-

Pseudouridine prevents the immune system from destroying the mRNA before it can deliver its therapeutic instructions. Substituting uridine with pseudouridine reduces the activation of innate immune receptors and significantly increases protein production.), thus helpful in RNA therapeutics.

Unmodified mRNA activated antiviral defences:

Dimerisation and phosphorylation

Activation of eIF2a

Inhibition of translation

Integrated stress response

Use of pseudouridine to prevent immunogenicity and increase translation

It was found that incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability

Scientists:

•Monitored IFN-alpha by ELISA

•Luciferase and GFP reporter mRNAs to measure protein output

Found capREN luciferase (it has a 5’ cap) to be more effective at increasing translational capacity than Tevluca (which doesn’t).

Used:

•luciferase reporter mRNAs to measure protein output

•Monitored PKR activation by wb and ³²P labelling of phospho-PKR

•Used PKR KO cells and viral PKR inhibitors to implicate PKR

Some advantages of RNA as a medicine include:

•speed (pathogen response/ personalized medicine) 

•Safety (no pathogen! No genetic change)

•local production

•Single optimized production workflow

New ideas to maximize gene expression

Optimise linear mRNA- with UTR design and optimisation, codon selection, structure stability optimisation,

Circular RNAs-

•Natural circRNAs are generated by ‘back splicing’-

Back-splicing is the primary molecular mechanism that generates circular RNAs (circRNAs) by covalently joining a downstream 5' splice site (splice donor) to an upstream 3' splice site (splice acceptor) in a reversed order compared to linear pre-mRNA splicing

• Can act as a sponge for miRNAs, RNA-BPs

• Highly stable

• Can be translated using IRESes or other cap-independent mechanisms

Self-amplifying RNAs-

•encode viral RNA-dependent RNA polymerase to replicate the RNA

Beyond mRNA- RNAs to disrupt gene expression

•First siRNA drug approved in 2018

•miRNA drugs in progress