In Vitro Synthesis of mRNA
The major components of mRNA are 5’-cap, 5’-UTR, open reading frame (ORF), 5’-UTR, and 5’ poly A tail, which are essential for maintaining mRNA function. Researchers have used a variety of methods to identify and optimize mRNA sequences and structures.
The synthesis of mRNA is performed on the basis of in vitro transcript (IVT) using linear DNA templates, RNA polymerases (T3, T7, or SP6), unmodified or modified nucleotides, enzymes, and appropriate reagents.
5’ Cap Modification
The sequences of mature mRNA from eukaryocyte show a 7-methylguanosine (m7G) cap at the 5’ end, which improves mRNA stability and translational efficiency. There are two general methods for capturing mRNA in vitro. First, mRNA can be capped along with in vitro transcript by adding a cap analog of the m7GpppG structure (e.g., CleanCap) to the IVT system. This co-transcriptional capping method provides a natural 5' capsule structure and increases the capping efficiency to nearly 90-99%. Secondly, mRNA mapping can also be accomplished by mapping enzyme reactions following the in vitro transcription reaction.
PolyA Modification
Poly(A) tail also extends the half-life of mRNA in vivo and improves mRNA translation efficiency. The length of the amplified poly(A) tail should be 100-300 nucleotides. Additionally, modified adenosine increases the stability of the poly A tail against cellular RNase degradation. Poly A tail can be inserted by in vitro transcription using DNA template encoding poly A, thereby resulting in a specific poly A sequence length. Recombinant poly A polymerase can also be used by enzymatic polyadenylation after mRNA transcription.
Nucleotides Modification
Modified nucleosides can inhibit pattern recognition receptors (PRR) recognition and /or activation and enhance the efficacy of mRNA vaccines in two totally different ways. he addition of certain chemically modified nucleosides including pseudouridine (ψ), 1-methylpseudouridine (m1ψ), thiouridine (s4U) and 5-methylcytosine (m5C) can prevent the activation of TLR7/8 and other innate immune receptors, which significantly reduce the immunogenicity of mRNA.
mRNA Delivery System
To maintain the function of mRNA, it needs to enter the host cytoplasm and express specific antigens. One of the most difficult challenges facing mRNA vaccines and therapeutics lies in delivering mRNA into target cells with sufficiently high translation levels for it requires highly specific and efficient mRNA delivery systems. Several mRNA delivery vectors have been developed and used, including dendritic cells (DCs), protamine, cationic polymers, and cationic liposomes.
Complexes of cationic lipids with mRNA and other preparations can collectively form 80-200 nm-sized nanoparticles named lipid nanoparticles (LNPs). As one of the most advanced mRNA delivery systems, LNP includes ionizable cationic lipids, natural phospholipids, cholesterol and polyethylene glycol (PEG). Several RNA vaccines and therapies (siRNA and mRNA) approved by the U.S. Food and Drug Administration are based on LNP delivery systems.
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Custom Deliverables
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Grade
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Deliverables
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Specification
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Applications
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non-GMP
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Drug Substance, mRNA
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0.1~10 mg (mRNA)
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Preclinical research such as cell transfection, Analytical method development, Pre-stability studies, Formulation development
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Drug Product, LNP-mRNA
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GMP, Sterility
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Drug Substance, mRNA
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10 mg~70 g
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Investigational new drug (IND), Clinical trial authorisation (CTA), Clinical trial supply, Biologic license application (BLA), Commercial supply
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Drug Product, LNP-mRNA
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5000 vials or pre-filled syringes/ cartridges
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