The main challenge in RNA and other nucleic acid-based therapeutics is how to deliver genetic information to target cells in the body. This barrier is currently being addressed, partly due to significant advancements in lipid nanoparticle (LNP) delivery systems. Successful cases of RNA-LNP include: the small interfering RNA-LNP drug Onpattro (patisiran) for treating hereditary transthyretin amyloidosis (hATTR), the COVID-19 LNP mRNA vaccines (notably the two vaccines from Pfizer and Moderna), as well as Moderna’s respiratory syncytial virus (RSV) vaccine mRNA-1345 and Arcturus’s self-amplifying RNA (saRNA) vaccine.
The key components of LNPs include ionizable cationic lipids, PEG lipids, phospholipids, and cholesterol. The intracellular delivery efficiency of LNPs is the main goal of their development. At the same time, as a new excipient or starting material, the potential safety issues of LNPs are also a major concern.
This article will focus on five popular RNA-LNP products that have been approved by regulatory authorities, analyzing their LNP lipid components and the ratios of each lipid (detailed in the table at the end of the article). These five products are Patisiran (siRNA-LNP), BNT162b2 (Pfizer/BioNTech mRNA COVID-19 vaccine), mRNA-1273 (Moderna mRNA COVID-19 vaccine), mRNA-1345 (Moderna RSV vaccine), and ARCT-154 (Arcturus/CSL saRNA COVID-19 vaccine).
Cationic Lipids
Ionizable Cationic Lipids (ICLs) are one of the key components of lipid nanoparticles (LNPs). Through electrostatic interactions, they bind with negatively charged nucleic acids (such as mRNA or siRNA), forming stable complexes. In addition, ionizable cationic lipids can be protonated in the acidic environment of endosomes, carrying a positive charge, which helps facilitate the escape of LNPs from endosomes and the release of nucleic acids into the cytoplasm.
Cationic lipids are the largest component of LNPs (accounting for approximately 50% molar ratio) and represent a major area of innovation for RNA-based therapeutics. The cationic lipid components of the five popular RNA-LNP products are detailed as follows:
- Patisiran (Onpattro, Alnylam): Uses the cationic lipid DLin-MC3-DMA.
- BNT162b2 (Comirnaty, BioNTech/Pfizer): Uses the cationic lipid ALC-0315.
- mRNA-1273 (Spikevax, Moderna): Uses the cationic lipid SM-102.
- mRNA-1345 (mRESVIA, Moderna): Uses the same cationic lipid as mRNA-1273, SM-102.
- ARCT-154 (CSL/Arcturus/Meiji): Uses the cationic lipid ATX-126.
With the approval of the first RNA-based drug using LNP technology, the cationic lipid MC3 has successfully reignited interest in RNA delivery. Various LNP components have been developed, accelerating the clinical development of other LNP-based RNA therapies, particularly mRNA COVID-19 vaccines. In less than a year, two LNP-based mRNA vaccines (mRNA-1273 and BNT162b2) were developed and deployed for large-scale clinical use at an unprecedented speed.
Interestingly, the cationic lipid component SM-102 in Moderna’s mRNA-1273 and ALC-0315 in BioNTech/Pfizer’s vaccine share common features, including tertiary amines, ester linkages, and branched tails. Additionally, both SM-102 and ALC-0315 have extended aliphatic branches, represented as multi-tail structures in the structural diagrams.
Moreover, several RNA vaccines/therapeutics delivered via LNPs are in clinical development, involving novel cationic lipids, as shown in the diagram below:
PEG Lipids
The primary function of polyethylene glycol-modified lipids (PEG-Lipids) is to increase the hydrophilicity and steric hindrance of lipid nanoparticles (LNPs) through the modification of the polyethylene glycol (PEG) chain. This modification promotes LNP self-assembly and prevents particle aggregation during preparation and storage, thereby enhancing particle stability. In addition, PEG facilitates the non-specific binding of LNPs to plasma proteins, extending their circulation time in the body.
PEG lipids make up the smallest proportion in LNPs, with a molar ratio ranging from 1.5% to 3%. The commonly used PEG lipid in RNA-LNPs is PEG2000-dimyristoyl glycerol (PEG2000-DMG), found in the following four RNA-LNP products: Patisiran, mRNA-1273, ARCT-154, and mRNA-1345.
In the development of BNT162b2, BioNTech/Pfizer used a novel PEG lipid: ALC-0159, chemically known as polyethylene glycol-distearoyl ethylamide (mPEG-DTA).
Lipids at BOC Sciences
- Branched Lipid
- Cationic Lipid
- Cholesterol
- Click Reagents
- Coenzyme A & Derivatives
- Enantiomers of Natural Lipids
- Fluorescent Lipids
- Formulations
- Ionizable Lipid
- Lipidomics
- Natural Lipids
- Other Lipid
- Phospholipid
- Polymers & Polymerizable Lipids
- Stable Isotopes & ESR Probes
- Sterols
Phospholipids
Phospholipids are essential components of the bilayer membrane structure of lipid nanoparticles (LNPs), providing membrane stability and fluidity in conjunction with cholesterol. Phospholipid molecules have hydrophilic heads and hydrophobic tails, which help form a stable bilayer membrane structure.
Phospholipids make up approximately 10% of the composition in LNPs. DSPC (distearoylphosphatidylcholine) is one of the phospholipid components found in several RNA-LNP products, including Patisiran, BNT162b2, mRNA-1273, mRNA-1345, and ARCT-154.
Cholesterol
Cholesterol plays a crucial role in regulating membrane fluidity, stability, and encapsulation efficiency in lipid nanoparticles (LNPs). By filling the gaps between lipids, cholesterol enhances the stability of nanoparticles. During cellular uptake, it helps facilitate fusion with the endosomal membrane, which aids in cellular internalization and improves the efficiency of nucleic acid delivery.
All five representative RNA-LNP products (Patisiran, BNT162b2, mRNA-1273, mRNA-1345, and ARCT-154) contain cholesterol as a component, making up approximately 40% of the total lipid content.
Summary
According to the GlobalData database, more than 1,000 RNA-based drugs are currently under development globally, with applications spanning oncology, central nervous system disorders, cardiovascular diseases, musculoskeletal conditions, as well as infectious diseases like HIV and hepatitis. These efforts aim to improve the treatment landscape for various diseases. Among these, lipid nanoparticle (LNP) systems that deliver RNA to target sites play a crucial role. However, LNP lipid components are under dual pressure from patent technologies and regulatory constraints, making the development of safe, innovative LNPs essential.
This article summarizes the lipid components and their proportions in five representative RNA-LNP products and compares the differences in their formulations to provide a reference for LNP development.
- Patisiran (Onpattro, Alnylam), a representative RNA-LNP product for small interfering RNA (siRNA), contains the following LNP components: cationic lipid DLin-MC3-DMA, phospholipid DSPC, cholesterol, and PEG2000-DMG.
- BNT162b2 (Comirnaty, BioNTech/Pfizer), the COVID-19 vaccine, contains the following LNP components: cationic lipid ALC-0315, phospholipid DSPC, cholesterol, and PEG lipid ALC-0159.
- mRNA-1273 (Spikevax, Moderna), the Moderna COVID-19 vaccine, contains the following LNP components: cationic lipid SM-102, phospholipid DSPC, cholesterol, and PEG2000-DMG.
- mRNA-1345 (mRESVIA, Moderna), the RSV vaccine, has the same LNP components as mRNA-1273.
- The first saRNA vaccine approved by PMDA, ARCT-154 (CSL/Arcturus/Meiji), contains the following LNP components: cationic lipid ATX-126, phospholipid DSPC, cholesterol, and PEG lipid ALC-0159.
| RNA Product | Ionizable cationic lipids | Phospholipids | Cholesterol | PEG lipid | Lipid molar ratio |
| mRNA-1273, Spikevax (Moderna) | SM-102 | DSPC | Cholesterol | PEG2000-DMG | 50:10:38.5:1.5 |
| BNT162b2, Comirnaty (BioNTech/Pfizer) | ALC-0315 | DSPC | Cholesterol | ALC-0159 | 46.3:9.4:42.7:1.6 |
| ARCT-154 (CSL/Arcturus/Meiji) | ATX-126 | DSPC | Cholesterol | PEG2000-DMG | 50:7:40:3 |
| mRNA-1345, mRESVIA (Moderna) | SM-102 | DSPC | Cholesterol | PEG2000-DMG | NA |
| Patisiran, Onpattro (Alnylam) | DLin-MC3-DMA | DSPC | Cholesterol | PEG2000-DMG | 50:10:38.5:1.5 |