Advances in the Safety Evaluation of mRNA Drug Delivery Carriers: A Non-clinical Perspective
Introduction
Messenger RNA (mRNA)-based therapeutics are one of the most successful and promising drug modalities in modern medicine. In only a few months, the first mRNA vaccine BNT162b2 was approved on an emergency basis for COVID-19, which brought hope to the whole world that these medicines are powerful tools for tackling the current pandemic. Besides fast production times, other potential benefits of mRNA drugs include high safety compared to DNA-based therapeutics, full biodegradability through natural metabolic processes, and an intrinsic adjuvant effect. Naked mRNA is a highly unstable molecule, and prone to quick degradation by widely present nucleases. Therefore, special carriers are needed to enable efficient cellular uptake and release into the cytoplasm for translation while protecting the mRNA from degradation. Although these carriers are the key enablers of clinical efficacy, they are also the major cause of safety issues in mRNA medicines. The understanding of their safety risk and development of non-clinical evaluation strategy are the key steps for further clinical translation of mRNA therapeutics. In this review, we summarize the potential toxicity risk for the major classes of mRNA carriers, their preclinical evaluation, and current limitations and future perspective in establishing a comprehensive safety evaluation system for mRNA therapeutics.
Potential Safety Risks of mRNA Carriers
- Lipid Nanoparticles (LNPs)
Lipid nanoparticles are currently the most widely used delivery vehicles for mRNA vaccines and therapeutics. They are composed of helper lipids, cholesterol, polyethylene glycol (PEG)-modified lipids, and ionizable cationic lipids. Despite their effectiveness, several toxicity risks have been identified.
Immune-related adverse effects:
PEGylated lipids are used to provide LNPs with stability, but are immunogenic and can cause complement activation-related pseudoallergy (CARPA). Anti-PEG antibodies, both IgM and IgG, may arise from LNP administration, and can result in accelerated clearance of the drug and reduced efficacy, or hypersensitivity reactions and in rare cases anaphylaxis. Case reports and clinical trials have associated mRNA vaccines tozinameran and CX-024414 with myocarditis and pericarditis, an immune-mediated adverse event particularly among young males.
Inflammatory response:
Ionizable cationic lipids, used for mRNA encapsulation and endosomal escape, have been associated with acute inflammatory responses, such as pain and swelling at the injection site, fever, and a systemic cytokine storm (IFN-γ, TNF-α, IL-6, etc.). In fact, cationic lipids are known to be able to activate complement pathways and trigger a strong inflammatory cascade.
Other toxicities:
In addition to inducing an inflammatory response, cationic lipids are known to trigger ROS generation, resulting in oxidative stress, apoptosis and hepatotoxicity. Increased liver enzymes, leukopenia and thrombocytopenia, as well as hepatic accumulation in animal studies have been linked to the uptake of LNPs by Kupffer cells. Hematologic abnormalities were seen with systemic exposure.
- Exosome-Based Nanoparticles
Exosomes are endogenously secreted vesicles that range in size from 40-160 nanometers in diameter. Their biocompatibility, ability to penetrate biological barriers, and low immunogenicity make them ideal mRNA delivery vehicles. There are, however, safety concerns that need to be taken into consideration, such as neurotoxicity and reproductive toxicity and immunogenicity. Exosomes are a potential method of mRNA delivery that requires further preclinical study before clinical use.
- Inorganic Nanoparticle Carriers
The most investigated inorganic nanoparticles are the stable gold, silver, and iron oxide nanoparticles. However, organ toxicity (hepatotoxicity, nephrotoxicity, genotoxicity) has been a concern. The potential long-term biopersistence of inorganic nanoparticles has also called their chronic toxicity into question.
- Virus-like Particles (VLPs)
VLPs (Virus-Like Particles) are self-assembled protein structures that mimic the shape of viruses but lack genetic information. Their high potential for packaging and mRNA delivery have been promising as carriers.
Local reactions: Injection site pain, swelling, and erythema were commonly reported. They were associated with high local inflammation.
Systemic reactions: Fatigue, gastrointestinal issues, and headache have also been seen in preclinical and clinical trials.
Safety concerns were not significantly raised, though the immunostimulatory effects of VLPs need further assessment.
- Other Nanoparticle Carriers
Biomimetic nanoparticles: Functional nanoparticles encapsulated by natural cell membranes. They have immune escape and targeting benefits.
Polymeric nanoparticles: These carriers are based on cationic polymers, dendrimers or polysaccharides. In general, they are biocompatible and biodegradable but they may also elicit immunogenicity or produce cytotoxicity depending on the polymer chemistry and dose.
Preclinical Safety Evaluation of mRNA Carriers
- Biodistribution Studies
The distribution of mRNA carriers across organs is a critical determinant of safety. Common evaluation methods include:
Luciferase-encoding mRNA bioluminescence imaging. For example, BNT162b2 demonstrated fluorescence in injection sites and liver at 6 hours after injection, but not at 48 hours.
Isotope labeling of lipid components for tracking. Distribution demonstrated in liver, spleen, adrenal glands and ovaries.
Branched DNA signal amplification for quantitative detection in various tissues. Moderna’s mRNA-1273 vaccine was found to be present in several organs, including brain, heart, lungs and testes.
Analyses such as these can be used to predict organ-specific toxicities and to help to optimize dose.
Immunogenicity and Immunotoxicity
Given that cationic lipids and PEGylated lipids are inherently immunogenic, assessing immune activation is central to safety evaluation.
- Antidrug antibody assays measure humoral responses.
- Flow cytometry is used to analyze T-cell subsets and cytokine release.
- ELISA assays assess specific antibody responses.
For instance, murine studies of mRNA vaccines demonstrated induction of CD8+ T cells, confirming the immunostimulatory potential but also highlighting the risk of exaggerated immune responses.
General Toxicity Studies
Systemic safety information is obtained from standard repeated-dose toxicity studies conducted in rodents and non-human primates. Toxicity study endpoints comprise clinical observations, local injection-site assessment, body weight, hematology, urinalysis, coagulation, histopathology and monitoring of cardiovascular function. BNT162b2 preclinical safety assessments have shown transient fever, injection site erythema, leukocytosis, and increased spleen weight, but no signs of irreversible toxicities.
These findings underscore the dose-dependent and often reversible nature of carrier-related toxicities, but also highlight the importance of comprehensive toxicological profiling.
Conclusion
mRNA therapeutics have already demonstrated their great potential and advantages in drug development, providing a fast, flexible approach to developing drugs for infectious diseases, cancer, and other diseases. The safety of mRNA drugs themselves is relatively high. Due to the different carriers utilized in the development of drug delivery carriers, related safety concerns are to be expected. For LNPs, the primary safety concerns are the potential for liver toxicity from the cationic lipid component as well as potential immune activation caused by PEG lipids. For exosomes, the potential for immunogenicity, concerns about their stability, and the possibility of transmission of infectious agents are the primary safety concerns. Inorganic nanoparticles such as Fe3O4 and gold have the potential to cause organ-specific toxicities and their long-term fate in the body is not yet well understood. The safety of VLPs is also not yet well established, and potential concerns include their immunogenicity and the potential to cause inflammation. Polymer-based systems may have issues with biodegradability, toxicity, and the potential for long-term accumulation in the body.
To ensure their safety and efficacy in clinical use, these drugs require comprehensive preclinical safety studies, including studies on biodistribution, immunogenicity, and general toxicology. Currently, there is a lack of standardized evaluation methods for the safety of mRNA drug delivery carriers. It is necessary for international regulatory agencies to establish a scientifically based and standardized evaluation system. As research continues on carrier toxicity mechanisms, it is expected to improve the rational design of safer and more effective delivery platforms for mRNA therapeutics.