►mRNA-based therapy-future and challenges
There is no doubt that the continuous progress of mRNA technology in the future will enable it to be widely used in more fields, especially cancer immunotherapy and infectious disease vaccines. For example, mRNA-based pluripotent stem cell induction, mRNA nuclease-assisted design, and protein delivery technologies have begun to be used in drug development. In the next few years, emerging therapies derived from mRNA technology will achieve rapid development, such as (1) allergy tolerance therapy, which uses edited plasmid DNA and mRNA to regulate allergic reactions caused by T helper and Treg cells; (2) cancer immunotherapy: enhance or change the autonomous immune system, make it better discover and attack cancer cells, or use microRNA and mRNA to regulate immune-related genes, thereby activating immune responses to fight cancer cells; (3) genome engineering therapy or gene therapy, including transplanting normal genes or editing nucleic acid genome sequences efficiently and accurately to replace missing or defective genes, thereby correcting genetic diseases or promoting inactive beneficial mechanisms or pathways, giving them the potential to treat genetic diseases; (4) protein replacement therapy: mRNA drugs are considered to provide an effective alternative to gene therapy, especially protein substitutes, because they avoid the risk of genome integration and provide powerful transient expression.
It is worth mentioning that the recently emerging gene editing system-CRISPR/Cas genome editing is applied. CRISPR/Cas gene editing is widely used in the research of system development and evolution. However, in recent years, scientists have used Cas proteins from prokaryotes to treat infectious diseases based on the heritable and adaptive immune characteristics of CRISPR in its original prokaryotic expression. These proteins are harmful, but when they enter the human body, they will cause an immune response and produce specific antibodies [4]. In addition, CRISPR/Cas technology is also integrated into tumor cell molecular biology research, used to quickly and accurately edit the genome, construct gene mutations to study tumor-related genes, or knock out genes for tumor treatment research.
In addition, mRNA-based combination therapy has gradually become a method for treating malignant tumors. As mentioned earlier, traditional vaccines do not seem ideal for preventing repeated rapid mutations of viral infections. Using mRNA vaccines to develop preventive or therapeutic vaccines is the most effective way to contain the new crown COVID-19 epidemic. Vaccines developed using mRNA technology reflect their advantages in production as well as their safety and preventive or therapeutic effects in clinical trials. In the next ten years, self-amplifying or replicon RNA vaccines and non-replicating mRNA vaccines will be more widely used.
In addition to the active exploration of mRNA vaccines for infectious diseases, the use of mRNA in emerging therapies for tumors will also achieve milestone development in the next ten years. It is worth emphasizing that the development of in vitro transcription mRNA therapy is still in its early stages. How to obtain a large amount of synthetic mRNA that can be used for clinical treatment is still a challenge. Problems related to extracellular reactions also need to be solved. Effective delivery methods need to be improved continuously. The vaccine is protected from various enzyme disruptions or avoids drug degradation when crossing the cell barrier.
To overcome these challenges, including using in vitro mRNA technology to re-edit and transplant autologous T cells or dendritic cells from patients, while continuously improving methods for delivering mRNA vaccines. At present, biopharmaceuticals and technology industries are investing resources to try to solve the following problems:
Stability-related issues
As mentioned earlier, mRNA molecules have an average of 1,000-6,000 base pairs and a large molecular weight (4-5 × 105 Dalton), because mRNA is synthesized using enzymes. Compared with smaller sRNAs, mRNAs are relatively large, making them inherently unstable and difficult to manufacture. The methods used to modify oligonucleotides such as locked nucleic acid (LNA) and unlocked nucleic acid (UNA) chemistry are limited on mRNA-based products. Therefore, there is an urgent need for a technology that can inject these drug molecules into the human body while maintaining the stability of the drug molecules and promoting cell uptake.
Effective delivery methods
Although there are several delivery methods currently available
1.Lipid-based delivery: using lipids or their derivatives/similar compounds to form particles (LNP), which can be used for in vivo delivery of mRNA therapeutics and vaccines. LNPs are nanoscale particles composed of synthetic or physiological lipid materials. The advantage is that RNA encapsulated in LNP is less likely to be enzymatically degraded, and the encapsulation efficiency is relatively high and easy to produce. In addition, cells effectively deliver mRNA molecules into the cytoplasmic sol by phagocytizing LNP;
2.Peptide-based delivery: various peptides, especially those containing cationic amino acids (such as lysine and arginine), can act as delivery carriers and form better complexes between carriers and mRNA through electrostatic interactions. The formation of complexes reduces the possibility of enzymatic degradation;
3. Polymer-based delivery: Polymer materials (such as polyamines, dendrimers and copolymers) can be used to effectively deliver mRNA candidates. This carrier also has the advantages of preventing enzyme degradation and supporting intracellular delivery. The problem is that they are usually not very stable. Therefore, the structure of polymer materials needs to be modified to improve the stability and safety of candidate drugs. For example, adding lipid chains, hyperbranched groups and biodegradable subunits, which will increase the process flow;
4. Virus-like replicon particles (VRP): Encapsulate small activating RNA (saRNA) and virus replicon particles, and deliver the encoding to the cytoplasm. Once inside the cell, saRNA will self-replicate and express the specified antigen. The advantage of VRP delivery is high efficiency. However, it faces two major challenges. First, a special manufacturing process is required for large-scale production of VRPs. Second, the human body may produce antibodies against the viral vector;
5. Cationic nanoemulsion: Cationic lipids composed of nanoemulsions are also used for RNA delivery. These compounds use their hydrophobic and hydrophilic properties to stabilize the lipid characteristics of drugs or vaccines, because the chemical interactions on the surface of molecules can protect mRNA from enzyme degradation, thereby achieving stable delivery of molecules. And nanoemulsions can be generated by some simple techniques, such as vigorous stirring, ultrasound or microfluidics;
6. In the future, the complex internal structure of the next generation of LNP needs to be strengthened, and its physical stability needs to be further enhanced, such as solid lipid nanoparticles, nanostructured lipid carriers and cationic lipid nucleic acid complexes. By chemical means, the ability to control the location and time of drug delivery and release in the body can be further improved, providing more effective or/and safer treatment for various diseases.
Safety issues
The issue of mRNA vaccines possibly inducing interferon responses seems unclear, which may be the root cause of side effects related to inflammation and autoimmunity? Another potential safety issue may come from extracellular reactions.
At present, the European and American drug administrations have not issued specific regulations on mRNA drugs
It may be based on mRNA therapy belonging to a broad category of genetic immunogens or vaccines, so mRNA drug research and development is classified as advanced therapy medicinal products (ATMP) or gene therapy medicinal products (GTMP) by the European Medicines Agency (EMA). However, as mRNA products continue to emerge in the medical field, it is very necessary to establish more specific regulatory regulations to make their research and development smoother, at least in the safety and efficacy of clinical trials related to this type of drug research and development need clear regulations , Or according to the unique function of mRNA, modify the specifications formulated by DNA vaccines and gene therapy vectors for research and development of new drugs or vaccines based on mRNA?
It can be affirmed that RNA therapy has made significant progress in different fields, and many therapies are at different stages of clinical (pre) development. With the advancement of molecular biology and a large amount of human and material resources invested, mRNA therapy has become a reality. With more investment in this field, mRNA therapy will be pushed to new heights and become a highly anticipated new treatment for cancer, infection and genetic diseases.
