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28 Pharmaceutical Technology ® The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com Drug Delivery Companies with expertise in one or more of these areas can form partnerships for the efficient develop- ment of new therapies. For example, a startup or new entrant in the field could bring knowledge of a disease and work with one company which offers expertise in construct design, quality control (QC) method devel- opment, process development, scale up, and chemistry, manufacturing, and controls (CMC) support; and source a delivery system with another party. Alternatively, they may prefer to partner with a company that can offer all these capabilities. Challenges in the development of RNA therapies The following key challenges exist in developing mRNA-based therapeutics: • W hat ma kes a n ef fec t ive m R NA con st r uc t? How is this construct optimized for stabilit y, expression, and yield? • Tolerability and immunogenicity of mRNA is greatly dependent on the clearance of process- and product-related impurities. • LNP biodistribution is often broad and mainly directed to the liver. • An immune response appropriate to the desired application is required, either immune-silent or stimulatory. • A p pr o pr i a t e a n a l y t ic a l m e t h o d s f or d r u g substance and drug product characterization must be employed. • Process technologies are required that enable manufacturing of RNA and LNPs at scale and in a cost-effective manner, while maintaining quality levels and critical quality attributes (CQAs). • T her mos t abi l it y—s torage a nd d i s t r ibut ion currently depend on effective and reliable cold chain logistics. To meet these challenges and optimize the chance of success of an mRNA–LNP therapeutic, an integrated approach is essential; this is because both the mRNA and LNP components have biological activity, and also aggregate physico-chemical properties. Optimization of RNA payload Production of mRNA using in-vitro transcription (IVT) typically uses a plasmid DNA template (other forms of template can be used). There are many components in a suitable template, and considerable resources have been devoted to sequence engineering to explore these. Figure 1 indicates the key components in a template— there are many factors to consider beyond just the cod- ing sequence of the target: Codon optimization. etherna has its own propri- etary codon optimization algorithm to allow a coding sequence to be adapted in such a way that RNA integ- rity and stability increase. Figure 2 indicates the improvements in the capil- lary gel electrophoresis (CGE) profile (a cleaner profile with a clear single peak) in mRNA produced from a codon optimized construct versus the original, for both standard and modified uridine. Sequence optimization can also significantly in- crease the expression of mRNA, as illustrated in TARGET CODING SEQUENCE CAP 5'-UTR SS DC- LAMP 3'-UTR polyA CAP structure reduces mRNA degradation and impacts activation of innate immunesystem, License to industry-leading CleanCap ® technology fromTriLink 5'-UTR: strong impact on translation initiation & peak level expression Coding sequence: impact on translation and mRNA stability impact on innate activation 3'-UTR: impact on mRNA stability and duration of expression polyA 'tail' length: mRNA stability and translation initiation FIGURE 1. Key elements to consider in mRNA construct design. FIGURES COURTESY OF THE AUTHORS. Production of mRNA using in-vitro transcription typically uses a plasmid DNA template.