mRNA Synthesis
IVT (In vitro Transcription)

By diluting in nuclease-free water or mild buffers like Tris-EDTA etc.

There are several plasmids available commercially for in vitro transcription. Several factors influence the choice of vectors or plasmids like cost, design, application, expression system, sequence of the 5’ and 3’ UTR, length of the polyA tail, template generation method, size of the plasmid and mRNA insert, IP etc.

Yield of mRNA generally depends on several factors related to the IVT process and the desired mRNA molecule. Process development is generally required for optimization of the IVT mRNA yield using different IVT template plasmids, promoter sequence, optimized UTRs, gene sequence optimization, reaction temperature and time, template purity, length of polyA, purification method etc.

There is no direct relationship between the level of mRNA and proteins. Studies estimating transcripts and proteins revealed the importance of several other processes and factors beyond transcript concentration that contribute to establishing the expression level of a protein. Aspects such as mRNA concentration, half- life, secondary structure, translation rates, mRNA and UTR sequences, internal ribosome entry sites (IRES), translation rate modulation, non-coding RNAs, ribosome abundance, protein half-life, internal cellular conditions, protein processing and degradation, protein synthesis delay, protein transport etc. influence the correlation between mRNA and protein levels in a cell. Thus, the direct comparison between protein and mRNA abundances from the same location or from the same cell type may not be appropriate.

Several methods such Capillary electrophoresis, Agarose and Polyacrylamide gel electrophoresis, Analytical Size-exclusion chromatography, qRT-PCR, Dot Blot, Mass Spectrometry etc. are employed for quality analysis of in vitro transcribed mRNA.

Explained in question no. 5

Once mRNAs enter the cell, they are either translated to proteins, stored for later translation (translational repression), or degraded by endonucleolytic cleavage. All mRNAs are ultimately degraded at a defined rate as per the cellular needs and mRNA half-life.

Quite a few strategies alone or in combination such as plasmid template purity, capping, polyA tail length, ORF sequence optimization, UTR sequences, use of base analogs, purification strategy, mRNA quality and removal of contaminants are generally considered to improve stability, export and translation of IVT mRNA.

Exogenous or IVT mRNA is inherently immune-stimulatory, as it is recognized by a variety of cell surface, endosomal and cytosolic innate immune receptors. It is potentially advantageous for vaccination because in some cases it may provide adjuvant activity to drive dendritic cell (DC) maturation and thus elicit robust T and B cell immune responses. Enzymatically synthesized mRNA preparations contain double-stranded RNA (dsRNA) contaminants as aberrant products of the IVT reaction. dsRNA and sometime structural elements of ssRNA is a potent pathogen-associated molecular pattern (PAMP) that is sensed by pattern recognition receptors such as Toll-like receptor resulting in robust type I interferon (INF) production when delivered exogenously. INF up-regulates protein kinases and synthetases, leading to the inhibition of translation and the degradation of cellular and ribosomal RNA. Besides translation products of the IVT mRNA are also recognized as foreign and elicit immune responses, a strategy used in vaccines.

Efficient in vivo mRNA delivery is of therapeutic significance. Exogenous mRNA must cross the barrier of the lipid membrane in order to reach the cytoplasm to be translated to functional protein. mRNA uptake mechanisms seem to be cell type dependent, and the physicochemical properties of the mRNA complexes can profoundly influence cellular delivery and organ distribution. There are two basic approaches for the delivery of mRNA vaccines. First, loading of mRNA into DCs ex vivo, followed by re-infusion of the transfected cells; and second, direct parenteral injection of mRNA with or without a carrier like protamine liposome, cationic polymers, nanoparticles etc.

Genome engineering, gene silencing, genetic reprogramming are some of the preclinical applications of IVT mRNA e.g. CRISPR Cas9. Use of IVT mRNA in clinical applications like are cancer immunotherapy, vaccines against infectious diseases, allergy tolerization, protein replacement therapies, regeneration therapies etc. are currently being explored as alternative immunotherapeutic.