mRNA Design, Synthesis, and Characterization for Gene Editing
Weijun Chen, PhD, Director, RNA Technologies Lead, Intellia Therapeutics
Weijun Chen, PhD, Director, RNA Technologies Lead, at Intellia Therapeutics, discussed the importance of mRNAs as key components of gene editing systems and therapeutic strategies during his presentation at TIDES USA in San Diego. He touched on the role of mRNA in molecular biology using the example of the central dogma. RNA can be made using DNA as a template and can control protein translation. There are several therapeutic applications of mRNAs. For example, due to RNA delivery systems, mRNAs can be translated to replace some proteins that are malfunctioning in patients. mRNAs can be designed to be used as vaccines to prevent or treat a disease. In addition, mRNAs can be translated into DNA-cutting enzymes such as SpyCas9 for gene editing.
Chen continued his talk with a focus on mRNA structure and function. Before producing mRNA for therapeutic applications such as vaccines, one should know the essential elements of mRNA, which are 5’ cap, 5’ untranslated region (UTR), ORF, 3’ UTR and poly(A) tail. 5’ cap provides stability to mRNA by protecting it from 5’-3’ RNA decay pathways. 5’ cap is also involved in translation initiation and immune stimulation. The preinitiation complex scans the 5’ UTR for cap-dependent initiation. primary sequence and secondary structure of the 5’ UTR influence this scanning process. In the absence of a cap, the 5’ UTR recruits ribosomes for cap-independent initiation. An open reading frame is important to improve protein expression and mRNA stability. Since codon prevalence is not homogeneous among organisms, codon usage should be considered in the mRNA design. The 3’ UTR provides space for a variety of RNA-binding proteins that give the transcript a means of fate such as stabilization, destabilization, localization, decay, and so on. The 3’ UTR contributes to protein translation and stabilization. The poly(A) tail protects the mRNA from hydrolysis and interacts with the 5’ cap to form a closed-loop structure that allows ribosomes to be recycled for the next translation.
Chen also mentioned that modified nucleotides can be incorporated into mRNA during in vitro transcription (IVT). For example, modified nucleotides such as modified uridine can increase translation efficiency and reduce immune stimulation in vivo. Following the design principles, Chen discussed the production of mRNA. He said mRNAs of interest are generally long. For longer mRNA, the IVT reaction is used. IVT requires inputs such as plasmid as a template, RNA polymerase, NTPs, and pyrophosphatase. You can also add a cap and tail and purify the product. How and when to cap and tail mRNA depends on the purpose of the mRNA and the downstream applications. There are two types of capping. Co-transcriptional capping allows capped mRNA to be produced in a single step. In post-transcriptional enzymatic capping, the IVT step is followed by the addition of a cap. The latter method can improve the yield of capped mRNA.
mRNA Production Methods
Chen presented two methods of poly(A) tail addition. It is possible to add 3’ polyT during template design to obtain poly(A) tail during IVT. The second way is enzymatic polyadenylation using poly(A) polymerase. Enzymatic polyadenylation can simplify plasmid production but produces mRNA batches with variable poly(A) tail lengths. Chen also mentioned impurities and purification, noting that dsRNA impurities cause unexpected immune stimulation. He said that cellulose chromatography can remove dsRNA impurities from IVT reactions. He added that purification of mRNA after IVT improves performance. Commercial kits are available for small-scale production, while salt precipitation and HPLC are other options for purification. He emphasized that the appropriate purification method depends on the scale of production and downstream applications.
While talking about analytical characterization and release testing, Chen explained critical quality attributes including identity, content, integrity, purity, and safety. Identity is related to sequence confirmation via NGS or Sanger sequencing. For the assessment of concentration, UV or RT-qPCR can be used. Integrity means the percentage of intact mRNA and fragment RNA and can be determined via HPLC. DNA or plasmid impurities can be detected using immunoblot or qPCR.
Chen also mentioned analytical methods for quantifying poly(A) tail length and capping efficiency. Poly(A) tail length can be assessed by RNase T1 treatment and capture via polyT-coated beads followed by LC-MS analysis. Capping efficiency can be determined using probes complementary to the 5’ end of mRNA. The combination of capture via magnetic beads and RNase H treatment enables the separation of mRNA 5’ ends. LC-MS of the dissociated fragments enables quantification of the capping ratio.
Chen concluded his presentation by mentioning future challenges in mRNA production. He explained two different mRNA structures: self-amplifying RNAs and circular RNAs. Chen said that, unlike conventional mRNAs, self-amplifying RNAs are inherently larger and require efficient production and purification processes. Circular RNAs are relatively more stable but require additional production steps.