Module 5

mRNA Therapeutics & LNPs

mRNA therapeutics went from laboratory curiosity in 2005 to >5 billion-dose COVID-19 vaccines by 2022. Two technological breakthroughs made it possible: pseudouridine modification (Karikó & Weissman, Nobel 2023) that evades innate immunity, and ionisable lipid nanoparticles (LNPs) that deliver mRNA across cell membranes. This module covers both and the clinical programmes beyond COVID.

1. The Pseudouridine Revolution

Unmodified mRNA is detected by TLR7/8 and RIG-I as viral RNA, triggering interferon responses that limit translation and cause systemic inflammation. Karikó 2005 (Immunity) showed that replacing uridine with pseudouridine (ψU) or 1-methylpseudouridine (m1ψU) abolishes innate detection while preserving ribosomal translation — and actually enhances translation 4–10×. Both Pfizer-BioNTech and Moderna COVID-19 vaccines use m1ψU.

2. Ionisable Lipid Nanoparticles

LNPs are composed of four lipids: ionisable lipid (SM-102, ALC-0315, DLin-MC3), PEG-lipid (steric shielding), cholesterol (structural), and helper phospholipid (DSPC). The ionisable lipid is key: neutral at blood pH 7.4 (low toxicity, long circulation), protonated in endosomes at pH ~5.5 (promotes membrane fusion and endosomal escape). Tuning pKa (ideal ~6.4) is the critical formulation decision.

Simulation: Ionisable Lipid & ψU

Python

script.py46 lines

import numpy as np, matplotlib
matplotlib.use('Agg')
import matplotlib.pyplot as plt

# pH-responsive lipid: ionisable at endosomal pH 5.5, neutral at blood pH 7.4
pH = np.linspace(4, 9, 200)
pKa_SM102 = 6.5
pKa_ALC0315 = 6.1
pKa_MC3 = 6.4

def frac_protonated(pH, pKa):
    return 1 / (1 + 10**(pH - pKa))

fig, (ax1, ax2) = plt.subplots(1, 2, figsize=(14, 5), facecolor='#0a0a1a')
for ax in (ax1, ax2):
    ax.set_facecolor('#111827'); ax.tick_params(colors='#cbd5e1')
    for s in ax.spines.values(): s.set_color('#334155')
    ax.grid(True, color='#334155', alpha=0.3)

ax1.plot(pH, frac_protonated(pH, pKa_SM102)*100, color='#2dd4bf', lw=2.5, label='SM-102 (Moderna)')
ax1.plot(pH, frac_protonated(pH, pKa_ALC0315)*100, color='#fbbf24', lw=2.5, label='ALC-0315 (Pfizer-BioNTech)')
ax1.plot(pH, frac_protonated(pH, pKa_MC3)*100, color='#a78bfa', lw=2.5, label='DLin-MC3 (Onpattro)')
ax1.axvline(5.5, color='#dc2626', ls='--', label='Endosomal pH')
ax1.axvline(7.4, color='#60a5fa', ls='--', label='Blood pH')
ax1.set_xlabel('pH', color='#cbd5e1')
ax1.set_ylabel('% protonated (cationic)', color='#cbd5e1')
ax1.set_title('Ionisable lipid pKa tuning for endosomal escape',
              color='#5eead4', fontweight='bold')
ax1.legend(facecolor='#1e293b', edgecolor='#334155', labelcolor='#cbd5e1')

# Translation kinetics: modified nucleotide vs unmodified mRNA
t = np.linspace(0, 72, 200)
prot_uU = 100 * (1 - np.exp(-t/2)) * np.exp(-t/6)
prot_mU = 400 * (1 - np.exp(-t/2)) * np.exp(-t/12)
ax2.plot(t, prot_uU, color='#94a3b8', lw=2.5, label='Unmodified U mRNA')
ax2.plot(t, prot_mU, color='#2dd4bf', lw=2.5, label='Pseudouridine (Kariko)')
ax2.set_xlabel('Hours post-transfection', color='#cbd5e1')
ax2.set_ylabel('Protein expression (relative)', color='#cbd5e1')
ax2.set_title('Kariko 2005: pseudoU boosts expression, cuts innate response',
              color='#5eead4', fontweight='bold')
ax2.legend(facecolor='#1e293b', edgecolor='#334155', labelcolor='#cbd5e1')

plt.tight_layout()
plt.savefig('output.png', dpi=120, bbox_inches='tight', facecolor='#0a0a1a')
print('Ionisable lipid cationic at endosomal pH; escapes endosome via membrane fusion')
print('Pseudouridine-modified mRNA evades TLR7/8 & RIG-I sensing -> higher, longer expression')

Click Run to execute the Python code

Code will be executed with Python 3 on the server

3. Applications Beyond COVID

Personalised cancer vaccines (mRNA-4157, Moderna-Merck): neoantigen mRNAs specific to each patient’s tumour mutations. Phase 3 in melanoma.
Seasonal flu vaccine (mRNA-1010): broader coverage than recombinant.
Respiratory syncytial virus (RSV): mRNA-1345 approved in 2024.
Rare disease: mRNA-3927 (propionic acidemia) replaces missing mitochondrial enzyme.
In-vivo CAR-T: Rurik 2022 mRNA-LNP targeting T-cells to express transient anti-fibrotic CARs.

4. Self-Amplifying & Circular mRNA

Self-amplifying mRNA (saRNA) encodes an alphavirus replicase that copies the mRNA inside cells, achieving the same protein output from 10–100× lower dose. Circular RNA (circRNA, Wesselhoeft 2018) has no free 5′ or 3′ ends, extending half-life; Orna, Laronde, and Circio are startup programmes. Both aim to reduce dose, increase durability, and broaden access.

Key References

• Karikó, K. et al. (2005). “Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.” Immunity, 23, 165–175.

• Pardi, N. et al. (2018). “mRNA vaccines: a new era in vaccinology.” Nat. Rev. Drug Discov., 17, 261–279.

• Hou, X. et al. (2021). “Lipid nanoparticles for mRNA delivery.” Nat. Rev. Mater., 6, 1078–1094.

• Wesselhoeft, R. A. et al. (2018). “Engineering circular RNA for potent and stable translation.” Nat. Commun., 9, 2629.

Share:X Reddit LinkedIn

← Module 4 Module 6: CRISPR →