RNA is a hydrophilic, negatively charged macromolecule, and its ability to autonomously transmembrane is limited. Therefore, how to efficiently deliver mRNA to the cytoplasm and play a corresponding role is one of the key issues that limit the application of RNA therapy.
The wide range of applications of therapeutic mRNA requires efficient and safe delivery methods. An ideal delivery system can carry mRNA through the cytoplasmic membrane without being degraded by RNase, and can allow mRNA to be released into the cytoplasm for translation after entering the cell. At the same time, the delivery system also has its corresponding safety. In recent years, a variety of strategies have been used for mRNA delivery, and most of these methods are inspired by siRNA and plasmid DNA delivery technologies.
Difficulties in mRNA vaccine delivery
As a new type of therapeutic drug, mRNA has great potential in vaccine research. The mRNA vaccine needs to pass the multiple tests of the "extracellular barrier", "endosome escape" and "intracellular immunity" to reach the target site in the cell and ultimately play a role. Therefore, how to specifically deliver mRNA to target cells is currently a difficult problem.
- Extracellular barrier
mRNA molecules are very easily degraded by enzymes, so they need to be protected from RNA hydrolase (RNAse) in extracellular serum during systemic administration to ensure that mRNA can reach target cells smoothly.
- Inner body escape
When reaching the target cell, the vector carrying mRNA enters the cytoplasm through endocytosis, which is also the most common way for the vector to enter the cell. Endosomal escape means that mRNA needs to be released from the endosomal vesicles, and then combined with the host cell ribosome to be translated into antigen proteins, which are modified and secreted out of the cell to play a role. In endosomal vesicles, mRNA can be detected by Toll-like receptors (TLRs) and sent for degradation, so endosomal escape is essential for mRNA to reach the ribosome.
- Intracellular immunity
When foreign mRNA is delivered to the cytoplasm, it can be recognized by TLR3 and TLR7/8, or by activating retinoic acid-induced gene I-like receptors in the cytoplasm, thereby activating the natural immune system. At the same time, foreign mRNA can also activate the expression of pro-inflammatory cytokines such as interferon.
Classification of mRNA delivery systems
- Usinga carrier to inject mRNA into the body
Commonly used carriers include dendritic cells, protamine, micro-plasmid carriers, polymer carriers and inorganic nanomaterials. Lipid nanoparticle carriers are one of the most commonly used carriers for mRNA vaccines. The cationic polypeptide protamine has been proven to protect mRNA from being degraded by ribonucleosidase, but there are also data showing that it can reduce protein expression, which may be caused by the tight connection between mRNA and protamine. Cationic liposomes and macromolecules, such as dendrimers, have been widely used in the delivery of mRNA in the past few years, which is also based on the development of small interfering RNA (siRNA). Lipid nanoparticles (LNPs) carriers have become one of the most commonly used carriers for mRNA vaccines.
- InjectingmRNA directly into the body
The most commonly used method of injecting mRNA directly into the body is intradermal injection or intranodal injection. It is reported that repeated intranodal inoculation of mRNA can stimulate T cell responses by tumor-associated antigens, which can improve the patient's progression-free survival.
- Physical delivery method
Physical methods can also be used to allow mRNA to penetrate the cell membranes. This delivery method does not require mRNA to escape from endosomes, nor does it need to consider issues such as carrier degradation. However, the disadvantage of physical methods such as electroporation, microinjection, hydrodynamic injection, and microfluidic cell delivery is that it may cause cell death, and the scope of application is relatively small.
