Therapeutic mRNA is a novel class of biopharmaceuticals that can deliver genetic information to cells and tissues and induce the expression of desired proteins for therapeutic purposes. Therapeutic mRNA has shown great promise in various fields of medicine, such as vaccines, gene therapy, protein replacement, and regenerative medicine. Compared to other gene-based therapies, therapeutic mRNA has several advantages, such as high potency, low immunogenicity, transient expression, and easy modification. The preparation of therapeutic mRNA involves several steps, such as designing the mRNA sequence, synthesizing the mRNA by in vitro transcription, and delivering the mRNA to the target cells and tissues. Each step requires careful optimization and quality control to ensure the safety and efficacy of the final product.
Fig.1 Types of synthetic mRNA for therapeutic application.1
The design of mRNA for therapeutic purposes is a crucial step that determines the stability, activity, and immunogenicity of the mRNA molecules. Several factors need to be considered when designing mRNA, such as nucleotide composition, codon usage, secondary structure, and modifications.
Nucleotide composition affects the stability and activity of mRNA by influencing its interaction with cellular components, such as ribosomes, enzymes, and immune receptors. For example, high GC content can increase the stability of mRNA by forming more stable base pairs, but it can also reduce translation efficiency by impeding ribosome movement and increasing immunogenicity by activating Toll-like receptors (TLRs). Therefore, a balanced GC content is preferred for optimal mRNA performance.
Codon usage affects the translation efficiency and accuracy of mRNA by influencing the availability and recognition of transfer RNAs (tRNAs). Codon usage can be optimized by using codons that match the tRNA pool of the target cells, avoiding rare codons that may cause ribosome stalling or misincorporation, and avoiding codons that may trigger premature termination or frameshifting. Codon optimization can also reduce the immunogenicity of mRNA by avoiding motifs that resemble viral sequences or induce interferon response.
Secondary structure affects the stability and activity of mRNA by influencing its folding and unfolding dynamics, which can affect its susceptibility to degradation, accessibility to translation machinery, and recognition by immune receptors. Secondary structure can be predicted and optimized by using computational tools such as RNAfold and RNAdesign. Secondary structure optimization can enhance the stability and expression of mRNA by avoiding regions that are prone to degradation, such as hairpins and loops, and favoring regions that facilitate translation, such as stem-loops and pseudoknots.
Modifications are chemical alterations that can be applied to mRNA to improve its performance, such as nucleoside analogs, 5' cap analogs, poly(A) tail length, and 5' and 3' untranslated regions (UTRs). Modifications can enhance the stability and expression of mRNA by protecting it from degradation, enhancing its translation, and modulating its localization and trafficking. Modifications can also reduce the immunogenicity of mRNA by masking it from immune recognition and attenuating its inflammatory response.
Fig.2 The typical structure of synthetic mRNA. 2
Different mRNA design strategies have different advantages and disadvantages, depending on the therapeutic application and delivery method. For example, mRNA with high stability and low immunogenicity may be suitable for protein replacement therapy, while mRNA with moderate stability and high immunogenicity may be suitable for vaccine development. Therefore, a tailor-made approach is needed to design mRNA for specific therapeutic purposes.
In vitro transcription (IVT) is a widely used method for synthesizing mRNA using DNA templates and RNA polymerases. IVT can produce large amounts of mRNA in a short time with high fidelity and low cost. IVT can also introduce various modifications to mRNA, such as 5' cap, poly(A) tail, and nucleoside analogs, by using modified nucleotides or enzymes in the reaction.
| Components | Description | Function |
|---|---|---|
| DNA template | A linear or circular DNA molecule that contains the promoter sequence and the coding sequence for the desired mRNA | Provide the template for mRNA synthesis. |
| RNA polymerase | An enzyme that catalyzes the synthesis of RNA from a DNA template, such as T7, T3, SP6, E. coli, B. subtilis, or eukaryotic RNA polymerases | Perform the transcription reaction. |
| Nucleotides | The building blocks of RNA, such as ribonucleoside triphosphates (rNTPs) or modified nucleotides | Provide the substrates for RNA synthesis. |
| Buffer | A solution that provides the appropriate environment for the IVT reaction, such as salts, metal ions, detergents, or stabilizers | Maintain the optimal conditions for the RNA polymerase and the DNA template. |
Table 1. The basic components of IVT
There are several challenges and solutions for optimizing IVT efficiency, yield, and quality:
The current technologies and platforms for scaling up IVT production for clinical applications are mainly based on two approaches: batch and continuous. Batch approach involves the use of large-scale reactors, such as bioreactors or fermenters, to perform IVT in a single vessel with a fixed volume and a defined duration. Batch approach can produce high-quality and homogeneous mRNA, but it has limitations in terms of scalability, flexibility, and cost-effectiveness. Continuous approach involves the use of microfluidic devices, such as microreactors or microchips, to perform IVT in a series of small chambers with a continuous flow and a variable duration. Continuous approach can produce high-throughput and low-cost mRNA, but it has challenges in terms of quality control, stability, and reproducibility.
| Cat.No. | Product Name | Price |
|---|---|---|
| GTVCR-WQ001MR | IVTScrip™ pT7-mRNA-EGFP Vector | Inquiry |
| GTVCR-WQ003MR | IVTScrip™ pT7-VEE-mRNA-FLuc Vector | Inquiry |
| GTVCR-WQ002MR | IVTScrip™ pT7-VEE-mRNA-EGFP Vector | Inquiry |
| GTVCR-WQ004MR | IVTScrip™ pT7-VEE-mRNA-mCherry Vector | Inquiry |
| GTVCR-WQ005MR | IVTScrip™ pT7-VEE-mRNA-β gal Vector | Inquiry |
| GTVCR-WQ006MR | IVTScrip™ pT7-VEE-mRNA-CCL1 Vector | Inquiry |
The quality and functionality of mRNA are critical for its safety and efficacy as a therapeutic or prophylactic agent. Therefore, it is essential to establish and implement appropriate analytical methods and quality control protocols for mRNA products. The main aspects of mRNA quality and functionality that need to be assessed are purity, integrity, identity, and potency.
Purity refers to the absence or minimal presence of impurities or contaminants in the mRNA product, such as DNA template, RNA polymerase, nucleotides, salts, or endotoxins. Impurities or contaminants can affect the stability, activity, and immunogenicity of mRNA and may pose potential risks to the patient. Purity can be measured by various techniques, such as gel electrophoresis, high-performance liquid chromatography (HPLC), mass spectrometry (MS), or capillary electrophoresis (CE).
Integrity refers to the completeness and correctness of the mRNA molecule, such as the presence and structure of the 5' cap, the poly(A) tail, and the coding sequence. Integrity can affect the translation efficiency and accuracy of mRNA and may influence its biological activity and safety. Integrity can be measured by various techniques, such as reverse transcription polymerase chain reaction (RT-PCR), MS, or sequencing.
Identity refers to the confirmation and verification of the mRNA sequence and modifications, such as the nucleoside analogs, the 5' cap analogs, and the 5' and 3' UTRs. Identity can ensure the consistency and specificity of the mRNA product and may determine its therapeutic or prophylactic effect. Identity can be measured by various techniques, such as sequencing, MS, or hybridization.
Potency refers to the ability and capacity of the mRNA to induce the desired biological response, such as the expression of the target protein, the elicitation of the immune response, or the modulation of cell function. Potency can reflect the functionality and performance of the mRNA product and may predict its clinical outcome and benefit. Potency can be measured by various techniques, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, or cell-based assays.
Therapeutic mRNA is a novel class of biopharmaceuticals that can deliver genetic information to cells and tissues and induce the expression of desired proteins for therapeutic purposes. Therapeutic mRNA has shown great promise in various fields of medicine, such as vaccines, gene therapy, protein replacement, and regenerative medicine. The preparation of therapeutic mRNA involves several steps, such as designing the mRNA sequence, synthesizing the mRNA by in vitro transcription, delivering the mRNA to the target cells and tissues, and assessing the mRNA quality and functionality. Each step requires careful optimization and quality control to ensure the safety and efficacy of the final product. Therapeutic mRNA has shown great promise in various fields of medicine, such as vaccines, gene therapy, protein replacement, and regenerative medicine. However, there are still many unresolved issues and unanswered questions that need to be addressed before mRNA can be widely and safely applied in clinical settings.
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