Principles of mRNA Liposome Vaccine Design

Summary

As a non-viral vector with good safety and efficient properties, lipid nanoparticles (LNP) have been clinically applied in mRNA delivery. In the recently FDA-approved SARS-CoV-2 mRNA vaccine, LNPs have been proven to be very suitable for antigen presentation and enhanced immune stimulation to trigger effective humoral and cellular immune responses.

Despite the rapid development of the mRNA-LNP vaccine, there are still some problems and challenges that need to be carefully considered:

1. Easy degradation of mRNA
2. Large-scale production
3. Transport and storage of vaccines at ultra-low temperatures
4. Delivery effect of mRNA
5. High immunogenicity and triggering of unwanted immune response

mRNA-LNP Design

In general, the mRNA-LNP design consists of several elements:

1. mRNA sequence design and nucleotide modification selection
2. Optimization of LNP preparations to encapsulate and deliver the mRNA
3. Long-term storage

• mRNA sequence design and nucleotide modification selection
• Nucleotide modification

Nucleotide modification is considered to be the first and most important part of mRNA therapy. Since unmodified nucleotides in the cell are recognized by RNA sensors, which initiate the innate immune response, the mRNA is cleared and not translated. Modification of coding region and codon optimization improved protein expression efficiency.

• mRNA capping

Capping mRNA significantly improves translation efficiency since it enhances mRNA intracellular stability and translation efficiency via binding to eukaryotic translation initiation factor 4E (eIF4E) In vitro transcription (IVT) capping is commonly used, either at IVT (one-step) or the end of IVT (two-step), but it should be noted that mRNA can be reversed 5'-cap, resulting in rapid degradation and low translation efficiency.

• UTR selection

The selection of non-coding regions greatly affects mRNA degradation and translation efficiency. UTR features such as the 5'-end initiation codon and secondary structure may affect ribosome recruitment, scanning, and initiation codon recognition, which should be avoided during design. Overall, the 5'-UTR sequence is critical for protein expression, while the 3'-UTR sequence is more likely to affect mRNA half-life. For example, repeated stabilization of β-globin 3'-UTR and β-globin 3'-UTR is widely used to stabilize mRNAs.

• Open reading frame (ORF) design

ORF sequence design also affects mRNA translation efficiency and immunogenicity, including nucleotide modification and codon selection mentioned above, which are important for protein expression. Use more annexation codons and codons with higher tRNA abundance Another sequence optimization scheme is to increase GC content, which has been shown to increase mRNA stability and protein expression in vivo.

• Poly(A) tail

Poly(A) tail can increase mRNA stability by reducing RNA exonuclease activity. In addition, the Poly(A) tail binds to the Poly(A) binding protein (PABP), which recruits eIF4G and eIF4E, enhancing the affinity for the mRNA cap to promote efficient translation of the circular mRNA structure as well as the subsequent translation.

• LNP/liposome components
• Ionizable lipid

It promotes self-assembly into tiny (<100 nm) particles (Including head group, linker area, and tail structure). and allows endosomes to release mRNA into the cytoplasm. Proper selection and construction of lipid structures contribute to improving mRNA stability, reducing toxicity, altering endosomal escape, and allowing mRNA release into the cytoplasm[3].

• Polyethylene glycol (PEG)-lipid

PEG-lipids are usually less than 2.5% of the total formulation, which prevents the aggregation and fusion of LNPs and increases the half-life of formulations.

• Cholesterol and phospholipids

Cholesterol plays a stabilizing role, and naturally occurring phospholipids support lipid bilayer structures.

long-term Storage

The long-term storage and stability of the mRNA-LNP vaccine is a major consideration in the formulation design and has not yet been fully addressed due to its high clinical relevance and the process of vaccine transportation and packaging, which ultimately affects the price of the vaccine.

According to product requirements, it can be frozen at -20 or -80, or stored in liquid nitrogen. The key point is that under such conditions, adding an appropriate amount of sucrose or trehalose to mRNA-LNP can not only retain the physical and chemical properties of the particles but also maintain the effectiveness of delivery in vivo for three months.

Lyophilization of mRNA-LNP vaccines could also be an option and would require the addition of lyoprotectants.

References

1. Liu T; et al. Design Strategies for and Stability of mRNA-Lipid Nanoparticle COVID-19 Vaccines. Polymers (Basel). 2022 Oct 6; 14(19): 4195.
2. Kon E; et al. Principles for designing an optimal mRNA lipid nanoparticle vaccine. Curr Opin Biotechnol. 2022 Feb; 73: 329-336.
3. Pardi N; et al. mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov. 2018 Apr; 17(4): 261-279.