A major obstacle facing the application of exogenous molecule-mediated biotherapies such as mRNA is its immunogenicity. With respect to reducing the immunogenicity of synthetic mRNA molecules in vitro, BOC Sciences' platform offers fully validated and large-scale synthetic nucleotide modification strategies to fully support our customers' mRNA vaccine development programs.
The major impediments to the application of in vitro transcribed mRNAs for vaccine development are instability and immunogenicity. Unmodified mRNAs are recognized intracellularly by RNA signaling receptors, which initiate the natural immune response, and not only is the mRNA cleared, but also causes serious side effects. Although the activation of natural immune response is beneficial in the field of vaccines, such as vaccine adjuvants, the expression of mRNA is also significantly reduced. Modification of nucleotides is a promising strategy for vaccine optimization for now, not only to reduce immunogenicity without affecting its translational properties.
Figure 1. Function of nucleotide modifications in mRNA.
Unmodified mRNA: Unmodified synthetic mRNA is unstable and stimulates activation of cellular innate immunity. Due to the high activity of environmental RNA enzymes, unmodified synthetic mRNAs are largely subject to degradation. Upon entry into the cell, unmodified RNA molecules are recognized by a variety of signaling receptors, such as the endosomal Toll-like receptor (TLR), cytoplasmic protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (2'-5'-oligoadenylase) oligoadenylate synthetase (OAS), retinoic-acid inducible gene I (RIG-I), or melanoma differentiation associated 5 (MDA5), and induces type I production of interferon, interleukin-6 and other pro-inflammatory cytokines, thereby stimulating the activation of cellular innate immunity.
Modified mRNA: Modified nucleotides evade the innate immune response mainly by blocking mRNA recognition by signaling receptors and avoiding activation of downstream signaling pathways. Pseudouridine reduces intracellular innate immunogenicity by blocking the stimulation of TLR7/8, PKR, OAS and RIG-I mediated signaling pathways. It is worth emphasizing that these cellular receptors sense not only the presence or absence of certain modifications, but also the RNA strand characteristics (single- or double-stranded RNA) and the location and combination of base modifications. m5C reduces intracellular innate immunogenicity by blocking stimulation of TLR7/8, PKR and RIG-1-mediated signaling pathways and stabilizes mRNA degradation, thereby prolonging half-life. m6A modification decreases the binding of TLR3/7/8, PKR, MDA5, OAS and RIG-I to mRNA, thereby evading innate immunity.
Based on research and existing experience, including Pfizer/BioNTech's BNT162b2, Moderna's mRNA-1273 and SARS-CoV-2 mRNA vaccine, nucleotide modification is now considered to be the most important breakthrough in mRNA therapy. CD BioSciences offers high-purity and high-quality nucleoside-modified mRNAs specifically developed for mRNA vaccines.
|The use of pseudouridine to completely replace uridine in mRNA not only greatly reduces the immunogenicity of mRNA, but also improves the stability and enhances the translation ability of mRNA.
|N1-methyl-pseudouridine is a methyl pseudouridine, and is a pseudouridine derivative with a methyl modification at the N1 position. It is a natural modification found in 18S rRNA and tRNA in many organisms. mRNA in which N1-methyl-pseudouridine enhances translation by an eIF2α-dependent and independent mechanism.
|The m6A modifications are related to mRNA stability, splicing processing, translation and microRNA processing.
|In mRNA, 5mC modifications along with various effector enzymes, such as NOP2/Sun RNA methyltransferase 2 (NSUN2), NSUN6,38 tRNA aspartate methyltransferase 1 (TRDMT1) and Aly/REF export factor (ALYREF), perform a variety of functions, including facilitating mRNA nucleocytoplasmic transport, viral protein expression, DNA damage repair, mRNA stability, cellular tolerance, proliferation and migration, stem cell development, and regulation of mRNA splicing.
|The addition of 5-methoxyuridine to RNA (mRNA) reduces the immunogenicity of the resulting mRNA. 5moU shows the highest level of protein production with negligible induction of inflammatory macrophage responses.
In addition to the above nucleoside modification services that specifically support mRNA vaccines, BOC Sciences offers additional RNA modifications. with a team of experts, high-end equipment and targeted strategies, BOC Sciences will be your best partner in the mRNA vaccine development process. We are available 24/7/365, please feel free to contact us.