In recent years, interest in RNA therapies has increased substantially. They are cost-effective to develop, relatively simple to manufacture, and can target conditions beyond the limitations of traditional small-molecule and macromolecular drugs.
Researchers are now developing mRNA and other small RNA drugs for a range of conditions, including cancer, neurodegenerative diseases, autoimmune diseases, metabolic disorders, respiratory diseases, and blood diseases. However, as their popularity increases, developers and sponsors must overcome numerous challenges to bring effective and safe mRNA therapies to market. These six key factors play a crucial role in the success of mRNA drug development.
1. Selecting the Best Delivery Systems for mRNA Therapies
The inherent instability factors of mRNA mean products need to be protected by specific packaging techniques and delivery systems. An effective means of delivery can reduce degradation by nucleases, increase absorption by target cells, and enhance the escape of endosomes.
Delivery systems must be chosen according to different indications and target tissues. Some of the options developers can consider include:
- Bare mRNA: can be administered through methods such as intramuscular injection, subcutaneous administration, and intradermal administration.
- Virus vectors: including the common adeno-associated virus (AAV).
- Polymers as carriers: include diethylaminoethyl glucose (DEAE), poly – β – amino ester (PBAE), polyethyleneimine (PEI), as well as degradable polymers such as polylactic acid glycolic acid copolymer (PLGA) and multifunctional copolymers such as dimethylaminoethyl methacrylate (DEAMA).
- Liposomes: including the popular lipid-based nanoparticle (LNP).
- Peptide carriers: such as shuttle peptide PepFect14, polymer peptide carriers, etc.
2. Deploying Effective Bioanalytical Strategies for mRNA Therapy Development
Bioanalysis is particularly complex in mRNA therapy development:
- mRNA therapies have a complex mechanism of action.
- Techniques must be cross-verified and validated.
- Multiple immunogenicity assays are often required.
- Few therapies have been approved, so there is little regulatory guidance.
Because mRNA products have a unique composition and varying mechanisms of action, several different and sometimes complex bioanalysis strategies must be deployed in the preclinical stage of development.
Biodistribution. toxicokinetic (TK), and pharmacokinetic (PK) studies are required to investigate each component of mRNA products, including the delivery system. However, if reliable safety data is already available for one part of the product, further testing on that component may not be necessary.
Biodistribution and PK studies may still be required in the clinical stage for mRNA vaccines when introducing new adjuvants, formulations, additives, or routes of administration.
If the mRNA expression product is a secreted or membrane protein, the distribution, existence, and metabolism need to be examined. For intracellular mRNA products such as anti-cancer vaccines, expression products are directly processed into peptide segments by antigen-presenting cells, so they don’t require detection and analysis. It’s also necessary to examine the immunogenicity of both the mRNA product and the expression product because both can independently trigger an immune response, and researchers must know which is causing the reaction.
To fully understand the safety and effectiveness of mRNA therapies, researchers must also assess immunotoxicity. In preclinical studies, this typically involves analyzing lymphocyte immunophenotyping and measuring cytokine secretion to detect any harmful immune responses. Other bioanalytical requirements include enzyme-linked immune spot detection (ELISpot) or flow cytometry-based intracellular staining to reveal the cellular immune response status from the cytokine levels produced by specific peptide segments and tracking special biomarkers related to toxicity and drug action mechanisms.
Both mRNA therapeutic products and mRNA vaccines require unique bioanalytical approaches. They are often best handled by a trusted lab partner with the expertise to overcome the many challenges that can arise.
3. Ensuring Stability and Translation Efficiency
Developers must tackle the stability and translation efficiency of mRNA products to ensure a smooth pathway through clinical trials. The most common strategies to improve stability include modifying the 5’ cap structure and 3’ polyadenylation tail, as well as the use of the Anti-Reverse Cap Analog (ARCA).
The regions of the mRNA that don’t code for proteins, the 5’ and 3’ untranslated regions (UTRs), also play a significant role in the stability of the mRNA and how effectively it is translated into protein.
4. Maintaining Immunogenicity Control
Studies have shown that nucleotide modification of exogenous RNA molecules can significantly reduce unwanted immune reactions in mRNA therapies and vaccines. Modifications such as m5C, m6A, m7G, inosine, and 2’-O-methylation may change the host immune responses and affect drug persistence and efficacy.
Other studies have shown that when mRNA includes bases such as 2-thiouridine or pseudouridine, it causes less cytokine release from dendritic cells and triggers a weaker immune response, allowing it to evade the body’s early defenses. Replacing the original uracil with N1-methylpseudouridine reduces immune reactions and boosts protein production.
5. Understanding Mechanisms of Action
Due to their differing delivery systems, administration routes, and types of expressed products, mRNA therapies and vaccines have a wide range of mechanisms of action. These include:
- Functional protein therapy based on mRNA: Products enter the cell through endocytosis and are translated and processed into target proteins. Common target proteins include antibodies, proteins or proteases, extracellular cytokines, and more.
- mRNA-based vaccines: After administration, the encoded protein is processed into small peptides by proteasomes, which are then transmitted to downstream immune systems by antigen-presenting cells.
- In vivo therapy: mRNA is delivered directly inside the body using LNPs designed to target T cells. They absorb the mRNA and produce chimeric antigen receptors (CARs), converting them into CAR-T cells that target and attack cancers.
- Gene correction and regulation: mRNA therapies can produce proteins that correct or regulate gene activity inside cells. For example, they can deliver instructions to make a DNA-modifying enzyme that locates and repairs faulty genetic sequences.
6. Navigating Regulatory Scrutiny
Major drug regulatory agencies have not yet developed comprehensive guidelines for the preclinical and clinical testing of mRNA therapies, making drug development in this area more complex.
Additionally, the analysis of mRNA components is primarily carried out using multiple platforms, including RT-qPCR, bDNA, ddPCR, and in situ hybridization (ISH) or Fluorescence in situ hybridization (FISH). However, there is also a lack of regulatory guidance for developing specific methodologies for the first three platforms.
Experienced lab partners can utilize their knowledge of global regulatory systems to guide developers and sponsors through this complex procedure.
A Final Word
The recent development and deployment of mRNA vaccines brought this technology to the world’s attention. These treatments can offer significant advantages over traditional biological therapies, including lower research costs, faster production, and stronger immune responses. The benefits are pushing mRNA therapies beyond infectious diseases to areas such as cancer treatment, enzyme replacement therapy, and preventive vaccines.
Many mRNA-based therapies are showing promising results in clinical trials, and with the correct development, they can offer patients more affordable and effective treatments in the future. To ensure therapies are as safe, effective, and delivered as quickly as possible, developers should work with lab partners who possess the tools and experience necessary to make a difference.
