In vitro transcribed (IVT) messenger RNA (mRNA) is a synthetic RNA molecule that can encode any protein of interest and deliver it to the target cells for expression. IVT mRNA has emerged as a promising tool for various biomedical applications, especially in the field of cancer immunotherapy and COVID-19 vaccination. IVT mRNA offers several advantages over other gene delivery methods, such as high efficiency, versatility, safety, and scalability. However, one of the major challenges of IVT mRNA therapy is its immunogenicity, which is the ability to induce immune responses in the host. Immunogenicity can be beneficial or detrimental, depending on the desired outcome of the therapy. For example, immunogenicity can enhance the antitumor or antiviral effects of IVT mRNA, but it can also cause unwanted side effects, such as inflammation, toxicity, or reduced efficacy. Therefore, understanding and modulating the immunogenicity of IVT mRNA is crucial for the development and optimization of IVT mRNA therapy.
IVT mRNA is recognized and sensed by the innate immune system, which is the first line of defense against foreign invaders. The innate immune system consists of various types of cells and molecules that can detect and respond to pathogen-associated molecular patterns (PAMPs), such as viral or bacterial nucleic acids. IVT mRNA shares some features with PAMPs, such as the presence of 5' triphosphate, double-stranded RNA (dsRNA), or unmethylated CpG motifs, which can trigger the activation of endosomal and cytoplasmic RNA sensors, such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), protein kinase R (PKR), and 2'-5'-oligoadenylate synthetases (OASes).
The activation of these RNA sensors leads to the initiation of downstream signaling pathways, such as the nuclear factor kappa B (NF-κB), interferon regulatory factor (IRF), and mitogen-activated protein kinase (MAPK) pathways, which result in the production of type I interferons (IFNs) and other pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12). These cytokines can have various effects and consequences on the host cells and tissues, such as inducing inflammation, apoptosis, necrosis, or pyroptosis, inhibiting protein synthesis, and enhancing antigen presentation and cross-presentation.
The immunogenicity of IVT mRNA can vary depending on the type and source of IVT mRNA, such as whether it is unmodified, modified, or purified mRNA. Unmodified IVT mRNA is more immunogenic than modified or purified IVT mRNA, as it contains more PAMP-like features and impurities, such as dsRNA, 5' triphosphate, and CpG motifs. Modified IVT mRNA is IVT mRNA that has been chemically or enzymatically altered to reduce its immunogenicity, such as by adding a 5' cap, optimizing the untranslated regions (UTRs), codon-optimizing the open reading frames (ORFs), modifying the poly(A) tail, or replacing the natural nucleosides with modified nucleosides, such as pseudouridine or N1-methylpseudouridine. Purified IVT mRNA is IVT mRNA that has been subjected to rigorous purification processes, such as high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE), to remove any dsRNA or other contaminants that may contribute to its immunogenicity. The immunogenicity of IVT mRNA can affect its safety and efficacy, as it can cause adverse reactions, such as fever, pain, or inflammation, or reduce its stability, translation efficiency, or expression duration.
One of the strategies to reduce or modulate the immunogenicity of IVT mRNA is to optimize the IVT reaction and purification processes, which can affect the quality and functionality of IVT mRNA. The IVT reaction is the process of synthesizing IVT mRNA from a DNA template using an RNA polymerase, such as T7, SP6, or T3. The IVT reaction can generate dsRNA byproducts or other impurities, such as DNA template, RNA polymerase, or nucleotides, which can increase the immunogenicity of IVT mRNA. Therefore, it is important to choose the appropriate RNA polymerase, reagents, and experimental conditions, such as temperature, time, and buffer, to minimize the generation of dsRNA or other impurities. IVT purification is the process of removing any dsRNA or other impurities from the IVT mRNA product using methods such as HPLC, PAGE, or affinity chromatography. The IVT purification can improve the purity, stability, and translation efficiency of IVT mRNA and reduce its immunogenicity.
Another strategy to reduce or modulate the immunogenicity of IVT mRNA is to modify the structure and composition of IVT mRNA, which can affect its recognition and interaction with the innate immune system. The structure and composition of IVT mRNA include the 5' cap, the UTRs, the ORF, the poly(A) tail, and the nucleosides. The 5' cap is a modified nucleotide that is added to the 5' end of IVT mRNA, which can protect IVT mRNA from degradation, enhance its translation efficiency, and reduce its recognition by RIG-I. The UTRs are the non-coding regions at the 5' and 3' ends of IVT mRNA, which can regulate the stability, translation efficiency, and localization of IVT mRNA and modulate its interaction with RNA sensors such as TLRs or PKR. The ORF is the coding region of IVT mRNA, which can encode the protein of interest and influence the immunogenicity of IVT mRNA, depending on the codon usage, GC content, and CpG motifs. The poly(A) tail is a string of adenine nucleotides that is added to the 3' end of IVT mRNA, which can protect IVT mRNA from degradation, enhance its translation efficiency, and modulate its recognition by OASes. The nucleosides are the building blocks of IVT mRNA, which can be natural or modified, such as pseudouridine or N1-methylpseudouridine, to enhance the stability, translation efficiency, and immune evasion of IVT mRNA.
A third strategy to reduce or modulate the immunogenicity of IVT mRNA is to use adjuvants, delivery systems, or immunomodulators, which can affect the uptake, distribution, and immunogenicity of IVT mRNA. Adjuvants are substances that can enhance the immune response to IVT mRNA, such as by increasing the antigen presentation, stimulating the innate immune system, or inducing the adaptive immune system. Delivery systems are vehicles that can facilitate the delivery of IVT mRNA to the target cells or tissues, such as by protecting IVT mRNA from degradation, increasing its uptake, or improving its biodistribution. Immunomodulators are agents that can modulate the immune response to IVT mRNA, such as by suppressing or stimulating the innate or adaptive immune system or inducing tolerance or immunity. Examples of adjuvants, delivery systems, or immunomodulators include transfection reagents, liposomes, nanoparticles, or cytokines.
Table 1. Comparison of strategies to reduce or modulate the immunogenicity of IVT mRNA.
| Strategy | Description | Advantages | Disadvantages |
|---|---|---|---|
| Optimizing the IVT reaction and purification processes | Controlling the synthesis and cleaning of IVT mRNA | Better quality and functionality, lower immunogenicity | Higher cost and complexity; need for standardization and validation |
| Modifying the structure and composition of IVT mRNA | Changing the features and components of IVT mRNA | Higher stability, translation efficiency, and immune evasion; lower immunogenicity | Possible impact on protein expression and folding; need for extensive optimization and testing |
| Using adjuvants, delivery systems, or immunomodulators | Adding substances, vehicles, or agents that affect the delivery and immunogenicity of IVT mRNA | Better uptake, distribution, and immunogenicity; desired outcomes of the therapy | Potential toxicity, immunogenicity, or interference; need for careful selection and combination |
Despite the significant progress and achievements in the field of IVT mRNA immunogenicity, there are still many gaps and limitations that need to be addressed and overcome. One of the challenges is the lack of standardized and validated assays, models, and criteria to measure and compare the immunogenicity of different IVT mRNA products and formulations. Currently, there is no consensus on the best methods and parameters to evaluate the immunogenicity of IVT mRNA, such as the type and dose of IVT mRNA, the route and frequency of administration, the duration and frequency of sampling, the choice and combination of biomarkers, and the interpretation and correlation of the results. Therefore, it is important to establish and harmonize the assays, models, and criteria to assess and compare the immunogenicity of IVT mRNA and to ensure the reproducibility, reliability, and relevance of the data.
With extensive research experience in the field of mRNA therapeutics, Creative Biolabs can provide customers with one-stop services such as IVT mRNA synthesis, mRNA modification, and quality assessment of mRNA products. Our aim is to collaborate with our clients to overcome obstacles in mRNA drug development.
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