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PharmTech.com The Real Message Behind Commercial mRNA Products April eBook 2023 Pharmaceutical Technology ® 29 Drug Delivery Figure 3 for the COVID-19 spike protein. Similar ef- fects have been seen in other constructs and have been previously reported (4). In this way, the codon adaptations allow for height- ened and prolonged expression, which may enhance the overall potency and efficacy of the mRNA ther- apeutic. As illustrated in Figure 3 for the COVID-19 spike protein, sequence optimization significantly increases the expression of mRNA. Similar effects have been seen in other constructs. Capping. Capping of the mRNA is important to minimize degradation and reduce the innate immune response. This can be done with standard capping technologies (e.g., ARCA for Cap0 capping or Vaccinia for Cap1, but generally we recommend proprietary capping technologies (e.g., CleanCap by TriLink BioTechnologies). The authors have seen processes optimized to achieve significantly high capping efficiencies (>95%) with low material use, thus reducing product cost. Optimized UnTranslated Regions (UTRs) for maxi- mum expression. By screening large libraries of 5' and 3' UTRs, mRNA made using etherna's proprietary tem- plate has increased expression (compared to internal and external benchmarks) by increasing intracellular expression. Figure 4 illustrates how well-designed and carefully paired UTRs can significantly increase ex- pression. Poly A tail. The poly A tail should be optimized to ensure the stability and integrity of the final product. Typically, the poly A tail is encoded in the plasmid template; although, it can be added enzymatically post-transcription. mRNA process optimization The in-vitro transcription (IVT) reaction can be op- timized in such a way that the formation of double stranded RNA (dsRNA) is drastically reduced, while maintaining high processing yields and low levels FIGURE 2. Indication on the improvements in the capillary gel electrophoresis (CGE) profile (a cleaner profile with a clear single peak) in mRNA produced from a codon optimized construct vs the original, for both standard and modified uridine. Relative expression measured by flow cytometry of cell surface anchored S spike all constructs delivered with identical eTheRNA LNP formulation FIGURE 3. Illustration of the significant impact of sequence optimization on expression of Covid Spike mRNA.