mRNA therapeutics are a novel class of biopharmaceuticals that use synthetic messenger RNA (mRNA) molecules to encode and deliver therapeutic proteins or induce gene editing in target cells. mRNA therapeutics have the potential to treat a wide range of diseases, such as infectious diseases, cancer, genetic disorders, and autoimmune diseases, by harnessing the natural cellular machinery to produce the desired proteins or modify the genome. Compared to conventional protein or gene therapies, mRNA therapeutics offer several advantages, such as high specificity, versatility, safety, and scalability. However, mRNA therapeutics also face several challenges and limitations, such as low stability, inefficient delivery, immune recognition, and off-target effects. Therefore, it is essential to develop and optimize methods and strategies to overcome these challenges and enhance the performance and efficacy of mRNA therapeutics.
The translation and stability of synthetic mRNA are crucial factors that determine the efficiency and duration of protein expression for therapeutic purposes. However, synthetic mRNA is subject to various degradation mechanisms and translational regulation in the cellular environment, which can reduce its potency and functionality. Therefore, various methods and strategies have been developed to enhance the translation and stability of synthetic mRNA, such as modifying the mRNA structure, cap structure, and nucleotide composition.
One of the most common and effective methods to enhance the translation and stability of synthetic mRNA is to modify the mRNA cap structure, which is the 5'-terminal m 7 GpppN (where N is any nucleotide) moiety that protects the mRNA from 5'-exonucleases and recruits the translation initiation factors. Synthetic mRNA can be capped in vitro by either enzymatic capping or co-transcriptional capping with cap analogs. Enzymatic capping involves the use of a capping enzyme complex that catalyzes the addition of GTP and the methylation of the 5'-terminal G. Co-transcriptional capping involves the use of cap analogs that are incorporated by the RNA polymerase at the 5'-end of the mRNA during transcription. Cap analogs can be modified at the 2'- or 3'-positions of the m 7 Guo or at the polyphosphate chain to improve their incorporation efficiency and resistance to decapping enzymes. For example, the anti-reverse cap analog (ARCA), which has a 3'-O-methyl group at the m 7 Guo, can increase the capping efficiency by preventing the reverse orientation of the cap analog. The boranophosphate cap analog, which has a boranophosphate group instead of a phosphate group, can increase the stability of the mRNA by inhibiting the decapping enzyme Dcp2. The phosphorothioate cap analog, which has a sulfur atom instead of an oxygen atom in the phosphate group, can also increase the stability of the mRNA by reducing its susceptibility to nucleases. Studies have shown that modified cap analogs can enhance the translation and stability of synthetic mRNA in both cultured cells and whole animals and increase the expression of therapeutic proteins or antigens.
Another method to enhance the translation and stability of synthetic mRNA is to modify the mRNA structure, which can affect the folding, accessibility, and interactions of the mRNA with various RNA-binding proteins and ribonucleases. Synthetic mRNA can be modified by altering the sequence, length, and composition of the untranslated regions (UTRs), the coding region, and the poly (A) tail. The UTRs play important roles in regulating mRNA localization, stability, and translation by containing various cis-elements that can bind to trans-factors, such as microRNAs, RNA-binding proteins, and ribosomes. The coding region can also influence mRNA stability and translation by affecting the mRNA secondary structure, codon usage, and initiation context. The poly (A) tail can enhance mRNA stability and translation by protecting the mRNA from 3'-exonucleases and facilitating the circularization and recycling of the mRNA. Synthetic mRNA can be optimized by using bioinformatics tools or experimental approaches to design the optimal sequence and length of the UTRs, the coding region, and the poly (A) tail, or by introducing modified nucleotides, such as pseudouridine and 5-methylcytidine, that can reduce the immunogenicity and improve the folding of the mRNA. Studies have shown that modified mRNA structure can enhance the translation and stability of synthetic mRNA in various cell types and tissues and improve the efficacy of mRNA-based vaccines and gene therapies.
The liver is a vital organ that performs various metabolic, detoxification, and immune functions and is also a major target for various diseases, such as viral hepatitis, liver cirrhosis, and liver cancer. Therefore, delivering mRNA therapeutics to the liver can offer great potential for treating these diseases, as well as for producing therapeutic proteins or inducing gene editing in hepatocytes. However, delivering mRNA to the liver faces several challenges, such as avoiding uptake by the reticuloendothelial system (RES), crossing the endothelial barrier, escaping from the endosomes, and achieving hepatocyte-specific targeting. Therefore, various non-viral vectors have been developed to overcome these challenges and enhance the liver-specific delivery of mRNA, such as lipid nanoparticles (LNPs), polymers, and ligands.
LNPs are one of the most widely used and successful non-viral vectors for liver-specific delivery of mRNA, as they can protect the mRNA from degradation, facilitate the endosomal escape, and mediate the cellular uptake by endocytosis. LNPs are composed of a lipid bilayer that encapsulates the mRNA, and can be modified with various components, such as ionizable lipids, helper lipids, cholesterol, and polyethylene glycol (PEG). Ionizable lipids are essential for the efficient encapsulation and release of mRNA as they can form electrostatic interactions with the negatively charged mRNA at low pH and release the mRNA at neutral pH. Helper lipids can improve the stability and fluidity of the lipid bilayer, and cholesterol can increase the rigidity and permeability of the lipid bilayer. PEG can increase the circulation time and reduce the immunogenicity of the LNPs by forming a hydrophilic layer that prevents the aggregation and opsonization of the LNPs. LNPs can also be modified with various ligands, such as galactose, or apolipoprotein E (ApoE), that can bind to the specific receptors on the hepatocytes, such as the asialoglycoprotein receptor (ASGPR) or low-density lipoprotein receptor (LDLR), and enhance the hepatocyte-specific targeting and uptake of the LNPs. Studies have shown that LNPs can deliver mRNA to the liver and induce the expression of therapeutic proteins or antigens, such as erythropoietin (EPO), factor IX (FIX), or luciferase, in various animal models, such as mice, rats, monkeys, and dogs.
Polymers are another type of non-viral vector that can deliver mRNA to the liver, as they can form stable complexes with the mRNA, protect the mRNA from degradation, and mediate the endosomal escape and cellular uptake. Polymers can be classified into natural or synthetic polymers and can be modified with various functional groups, such as amino, carboxyl, hydroxyl, or sulfhydryl groups, to adjust their physicochemical properties, such as charge, size, shape, and biodegradability. Polymers can also be conjugated with various ligands, such as peptides, antibodies, or carbohydrates, that can enhance the liver-specific targeting and uptake of the polymers. Some examples of polymers that have been used for liver-specific delivery of mRNA are polyethylenimine (PEI), poly (amidoamine) (PAMAM) dendrimers, poly (lactic-co-glycolic acid) (PLGA), chitosan, and hyaluronic acid. Studies have shown that polymers can deliver mRNA to the liver and induce the expression of therapeutic proteins or antigens, such as erythropoietin (EPO), factor IX (FIX), or hepatitis B surface antigen (HBsAg), in various cell lines and animal models, such as HepG2 cells, mice, and rats.
