Messenger RNA (mRNA) is a type of single-stranded RNA involved in protein synthesis. Its role is to transfer genetic information from DNA to ribosomes, provide the amino acid sequence of the protein required for DNA coding, so that the cells in the patient's body become "in vivo factories" to produce drug therapies, and guide the human body cells to produce intracellular or secretory proteins that have therapeutic or preventive benefits for both the patient and the healthy individual.
The great success of the mRNA COVID-19 vaccines have revived interest in using mRNA to express therapeutic proteins. In addition to the mRNA COVID-19 vaccine, a series of clinical trials have begun using mRNA to express vascular endothelial growth factor (VEGF) to treat heart failure, and CRISPR-Cas9 mRNA to treat rare genetic diseases. However, a number of challenges remain to be addressed before mRNA can be established as a universal therapeutic modality for rare and common diseases. To overcome these challenges, scientists are developing a series of new technologies, including optimization of mRNA sequences, development of organ/tissue-specific lipid carriers, and in vivo transdermal drug delivery systems. The combination of these advances holds the promise of unlocking the promise of mRNA therapeutics beyond vaccines to treat a variety of disease types.
A review paper entitled: Unlocking the promise of mRNA therapeutics was recently published in Nature Biotechnology, discussing how to unlock the promise of mRNA therapeutics in terms of mRNA design and purification, improving the timing and level of mRNA expression, improving mRNA delivery systems, tissue-specific delivery systems, and repeat drug delivery strategies, and summarizing current clinical trends in mRNA therapeutics.
The widely proven safety and efficacy of mRNA COVID-19 vaccines, which have been administered in billions of people around the world, suggests the potential to develop a new generation of mRNA-based therapies beyond vaccines.
Differences between mRNA vaccines and mRNA therapeutics
Compared with mRNA vaccines, the development of mRNA therapeutics faces more challenges. Because mRNA vaccines only need to produce a small amount of protein, the body's immune system amplifies the immune signal through cellular and antibody-mediated immune responses. mRNA therapeutics requires more than 1,000 times the level of protein expressed by mRNA vaccines to reach the therapeutic threshold.
Moreover, typically, mRNA therapeutics need to act on specific target pathways, cells, tissues or organs. Therefore, attention should be paid to the absorption of mRNA by target cells, which determines the duration and level of mRNA expression. The bioavailability, cycle half-life and delivery efficiency of lipid carrier delivery to tissues may be rate-limiting factors.
By intravenous injection, mRNA therapeutics can be easily targeted to the liver, but their effective delivery to other solid organs remains challenging. In addition, repeated administration is currently facing obstacles. For the treatment of chronic diseases, multiple administration is usually required, but even optimized mRNA and LNPs can activate innate immunity after multiple administration, thus reducing the expression of therapeutic monowhite.
F1 mRNA Vaccines and mRNA Therapeutics
1. Increase protein production
While mRNA's inherent immunogenicity enhances its effectiveness as a vaccine, it also hinders its potential as a therapy. mRNA therapy requires high levels of protein expression to achieve therapeutic effect, and in mouse models used for enzyme replacement therapy, local regenerative therapy, and tumor immunotherapy, doses 50 to 1000 times higher than those used for mRNA vaccines are typically required. The need for high protein expression levels has led to a variety of strategies for optimizing mRNA to minimize immune responses, enhance mRNA stability and maximize translation efficiency.
The figure below is a schematic diagram of different modifications of mRNA that are currently in clinical use or are being studied to improve protein expression efficiency. mRNA consists of five main domains - 5' cap, 5' untranslated region (5'UTR), open reading frame (ORF), 3' untranslated region (3'UTR) , Poly(A) tail (PolyA). Optimization of these five domains can enhance protein expression levels.
The innate immunogenicity of mRNA, while enhancing its effectiveness as a vaccine, hinders its use as a therapeutic agent that requires higher levels of protein expression. The need for high levels of protein expression has led to a variety of strategies to optimize mRNA load to minimize innate immune responses, enhance mRNA stability, and maximize translation (see Figure 2). However, for any given indication, the nature of the mRNA cargo must be related to the efficiency of the delivery system (e.g., direct versus systemic injection) and the mode of action of the protein of interest.
F2 Optimization of different mRNA structures
For mRNA vaccines and mRNA therapeutics, perhaps the most critical development is the discovery that chemical modifications to nucleosides can significantly reduce the immunogenicity of mRNA and increase protein expression levels. This is also at the heart of patent claims in the mRNA field so far. In addition to chemical modification of mRNA, codon optimization of mRNA sequence is also expected to develop effective therapeutic mRNA without chemical modification.
In addition to protein expression levels, a key limiting factor for mRNA therapeutics in treating chronic diseases is its short protein production time and therefore the need for repeated administration. There are several optimizations of mRNA structure to increase the duration of protein expression, such as self-amplifying mRNA (saRNA) and circular mRNA (circRNA).
Self-amplifying mRNA (saRNA) utilizes the self-replicating ability of RNA alphavirus, which can self-replicate in cells, thereby reducing the dosage and frequency of administration. Compared with linear and modified mRNA, only one-tenth the amount of self-replicating mRNA is needed to achieve similar protein expression levels. A number of self-amplifying mRNA COVID-19 vaccines are currently in clinical trials. In addition, there is another form of self-amplifying mRNA - trans-amplified mRNA (taRNA), which puts the replicase and the target gene on two mRNAs, which is safer and helps to reduce the size of the mRNA.
Circular mRNA (circRNA), which circularizes linear mRNA, can prevent mRNA from being degraded by exonucleases, extend the half-life of mRNA in cells, and increase its total protein expression. Moreover, circular mRNAs avoid the expensive 5' caps and cumbersome Poly(A) tails that linear mRNAs must add. Furthermore, circular mRNAs significantly reduced immune responses without chemical modification.
F3 Different mRNA forms
It should be noted that the purification of mRNA and the reduction of by-product double-stranded RNA (dsRNA) can not only reduce the immune response, but also increase the protein expression level.
2. Vectors And Delivery Systems
The inherent instability of mRNA leads to the need for a packaging/delivery system to protect mRNA from nuclease degradation and allow it to be taken up by the cell, released within the cell, and translated into proteins.
F4 Intracellular delivery and translation of mRNA
LNPs delivery system
Most of the mRNA vaccines/therapies currently on the market and in development use lipid nanoparticles (LNPs) as carriers. LNP was first proposed more than 60 years ago and has since undergone many changes and advances, finally being used clinically for the first time to deliver siRNA therapy. LNPs consists of four key components: structural lipids, cholesterol, cationic or ionizable lipids, and invisible lipids. Structural lipids are the basic scaffolds of LNPs, and the addition of cholesterol in different proportions can stabilize the structure of LNPs and regulate its properties, such as membrane fluidity, elasticity and permeability. Cationic lipids or ionizable lipids are essential for loading negatively charged mRNA into LNP. The invisible lipids are mainly polyethylene glycol (PEG) modified lipids, whose addition can reduce the immunogenicity and increase the stability of LNP. But it's worth noting that some people are allergic to PEG, which can be a recurring obstacle in the treatment of chronic diseases. Therefore, the optimization of polyethylene glycol (PEG) modified lipids or the development of other stealth lipids is the focus of current research.
F5 Development milestones of the LNPs
Meanwhile, other delivery vectors based on cells, extracellular vesicles and bionic vesicles are being developed and validated as alternative vectors in preclinical studies.
F6 Advantages and challenges of LNP, extracellular vesicles, cells and bionic vectors
3. Tissue Targeting
Realizing the full potential of mRNA therapy will require more advanced delivery systems in the body, especially for solid organs such as the heart, kidneys, brain and lungs. For most molecular therapies, the liver is the easiest organ for delivery, and its porous vasculature facilitates efficient uniform delivery and passage of large particles. Thus, simple intravenous administration enables efficient expression of mRNA cargo in the liver with corresponding therapeutic protein levels (Supplementary Table 1). However, targeting most organs other than the liver requires improved delivery systems, either directly through the catheter or through the engineering of appropriately oriented packaging systems. Each organ has its own advantages and barriers to efficient delivery.
4. Drug Administration For Chronic Diseases
The ability to specifically and efficiently deliver mRNA repeatedly while maintaining high protein yields is a key requirement for the transition of mRNA from vaccines to therapeutic drugs. Enzyme replacement therapy that relies on recombinant proteins illustrates this point vividly. For example, hemophilia A and B blood disorders due to a deficiency of the clotting protein are usually treated with 3–7 weekly systemic injections of factor VIII or factor IX recombinant protein, respectively, with a relatively short half-life of approximately 12 hours. Preclinical studies in mice have shown that this regimen can be replaced by a single weekly systemic injection of 0.2–0.5 mg/kg of linearly modified mRNA while maintaining protein levels above clinically relevant thresholds (Supplementary Table 1). In another approach, clinical results of DNA-based gene therapy for hemophilia using AAV vectors showed an increase in protein levels during the first 2 years, after which they leveled off. Recent data suggest that supplementation is required after 5-7 years due to immune rejection of viral vectors. Viral vectors have their own safety concerns, especially in pediatric indications.
The real added value of mRNA therapeutics compared to protein drugs is the ability to synthesize high levels of intracellular protein. This in vivo approach enables direct targeting of metabolic diseases such as Crigler-Najjar syndrome, methylmalonic acidemia, propionic acidemia, and cystic fibrosis, which are technically difficult to treat with proteins (Supplementary Table 1) . For example, current treatment of propionic acidemia consists of activating urea production of carboglutamate through ingestion of 100–250 mg/kg per day. Although it mitigated the toxic buildup of ammonia, it did not treat the underlying metabolic defects. In contrast, a dual dose of 0.5 -- 2 mg/kg − of hPCCA and one of hPCCB mRNA every 3 weeks in a knockout mouse model showed sustained reductions in plasma biomarker and enzyme activity for 3 months and is currently in Phase I clinical trials.Although it mitigated the toxic buildup of ammonia, it did not treat the underlying metabolic defects. In contrast, a dual dose of 0.5 -- 2 mg/kg − of hPCCA and one of hPCCB mRNA every 3 weeks in a knockout mouse model showed sustained reductions in plasma biomarker and enzyme activity for 3 months and is currently in Phase I clinical trials.
5. Clinical Research
mRNA vaccines have successfully completed phase III clinical trials and received international regulatory approval, while most mRNA therapeutics are in early clinical phase I studies with a major focus on safety (Supplementary Table 2: mRNA Therapeutics Clinical Trials). Given that mRNA therapeutics can produce virtually any protein either systemically or locally, a broad range of potential disease indications and protein classes is currently being investigated. Protein classes that can be delivered by mRNA include enzyme proteins, receptors, intracellular proteins, mitochondrial membrane proteins, secreted proteins, and gene editing proteins (Table 3: Summary of different classes of potential mRNA therapeutics). To date, only two clinical studies have produced encouraging results in terms of safety and efficacy signals: VEGF mRNA for heart failure and mRNA encoding CRISPR–Cas9 for hereditary amyloidosis.
Table 3: Summary of different classes of potential mRNA therapeutics
Thirty years of scientific and clinical progress, combined with enormous efforts to develop an mRNA COVID-19 vaccine, heralds a promising future for mRNA therapeutics. Today, we are able to rapidly design and synthesize clinical-grade mRNA in an automated, scalable, cell-free format with only a few mouse clicks.
In the near future, we also have the potential to generate modular, scalable GMP grade manufacturing units that can be located in any GMP grade facility, eliminating the need for cold chain transportation. Lyophilized stored mRNA therapeutics will also be available, which will largely solve the distribution problems of current mRNA vaccines due to the difficulty of transportation and preservation. With the development of new LNP and non-LNP vectors, the side effects will also be improved. Increased carrier capacity makes it possible to deliver complex genes and base editing, and with repeatable delivery, mRNA therapy is expected to replace current protein replacement therapies.
Looking back at the history of recombinant protein therapy, in the early days of the field, it was expected that most growth factors would become drugs. However, it remains to be seen whether VEGF will become a clinically valuable treatment now, 30 years after it was cloned. The future of mRNA therapeutics may therefore depend on matching this "software of life" to the "hardware" of the human physiological system, improving accuracy, extending duration under safety conditions, and delivering long-term chronic drugs.
In the coming years, the rapid development of mRNAs, intracellular vectors, and delivery systems in vivo, combined with in-depth biological and clinical insights and intuition, should offer new hope to many patients with clinical needs that cannot be easily met by other therapeutic modalities.
It is expected that with the advancement of science and the development of technology, mRNA therapeutics will be used more widely. As a reliable worldwide supplier of PEG & ADC linkers, Biopharma PEG supplies a variety of high purity PEG derivatives, PEG linkers and ADC linkers to empower drug research & development. We can produce and provide some PEG products as ingredients used in COVID-19 vaccines.
Eduarde Rohner , Ran Yang, Kylie S. Foo,et al, Unlocking the promise of mRNA Therapeutics, nature biotechnology, volume 40, 2022.10, 1586-1600
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