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16 Pharmaceutical Technology ® The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com mRNA TReNds nucleic acids, nucleoside triphosphates, and mRNA capping analogs. Henderson notes, however, that mRNA production scale-up, specifically, is quickly being overcome through optimized in-vitro transcrip- tion reactions followed by simplified purification processes. Optimal on-target delivery and standard- ization of drug substance analytical procedures, how- ever, are still a work in progress, she adds. "To make a functional message, it is critical that the synthetic mRNA contains all the structural elements of a mature post-transcriptionally modified mRNA found in eukaryotes. This includes a 5' cap structure (Cap 1 or 2), 5' untranslated region (UTR), coding sequence, 3' UTR, and poly-adenosine tail," Henderson explains. She also specifies that generating the appropriate 5' Cap 1 structure in vitro once required additional en- zymes and purification steps in the manufacturing process. Now, however, using co-transcriptional cap analogues (e.g., CleanCap, TriLink) can initiate the for- mation of a Cap 1 structure in a one-pot reaction via the RNA polymerase that is used to transcribe the message of interest, she further elucidates. "Such improvement to the IVT [in-vitro transcription] reaction has enabled streamlined and scaleable purifi- cation processes for quick manufacturing of high-qual- ity mRNA products. Ongoing and future work on the mRNA platform is focused on developing safe modifi- cations to the nucleic acid that increase the molecules stability and translatability," says Henderson. Henderson a lso points out that severa l groups, including academic and industry alike, are actively work i ng on l ipid n a nopa r t ic le del iver y s y s tem improvements, while regulatory leaders, including t he US Pha r macopeia l Convent ion a nd FDA, a re looking to standardize mRNA drug substance spec- if ications. "Altogether, these ef for ts will contrib- ute to much needed advances in medical treatment options," Henderson states. Becraft points to the specificity of therapeutic pro- tein expression as another major challenge in devel- oping nucleic-acid agents. By using synthetic biology, however, the industry can address this roadblock and improve safety and efficacy. "Synthetic biology cou- pled with mRNA guarantees that the molecule will be encoded with the correct genetic information. We can also engineer the mRNA to be context-dependent so the protein will only be expressed inside of certain cell types. This logic-based genetic programming al- lows us to have precise control of the location, timing, and level of protein expression," Becraft states. Lewis also hones in on the limitations in tolera- bility and immunogenicity, as well as scalability, of current delivery methods, as being among the major challenges to nucleic acid-based therapy develop- ment. "Lipid-based systems are associated with liver toxicities and viral-based systems lead to immune responses that can both limit initial transduction ef- ficiency and prevent redosing. Entos was established to address these limitations and broadly enable the next generation of nucleic medicines," he states. "We are in the process of publishing preclinical data demonstrating the significant potential of our Fusogenix proteolipid vehicle (PLV) platform in di- verse indications. These data show that this novel delivery system leads to broad distribution across tis- sue types and organ systems with minimal toxicity. Gene expression can be targeted using tissue-specific promoters, and delivery of DNA with the Fusogenix PLV platform [e.g., Entos' DNA vaccine candidate] leads to sustained gene expression out to one-year post-dosing," Lewis says. The data from Entos' as-yet-unpublished preclini- cal studies also demonstrate that the Fusogenix PLV platform can effectively deliver RNA as well. "Impor- tantly, these studies also show that the low levels of immunogenicity and high levels of activity are main- tained after extended periods of repeat dosing," says Lewis. "Additional studies demonstrate the feasibil- ity of using the Fusogenix platform to enable DNA vaccines against SARS-CoV-2." Entos' DNA vaccine candidate includes the f ull- length viral spike protein, which may provide en- hanced and broad immune responses compared with protein vaccines that use spike protein fragments, Lewis adds. DNA vaccines can also encode genetic adjuvants that can enhance neutralizing anti-SARS- CoV-2 antibody responses in mice and non-human primates. "Data demonstrates that our Fusogenix PLV DNA vaccine candidate protected against SARS- CoV-2 infection in an animal model. We are currently conducting a Phase II trial of our SARS-CoV-2 DNA vaccine candidate,"Lewis states. Historically, the delivery of oligonucleotides into t he cel l has been a n issue for R NA t herapeut ics, says Dispersy n. Companies have ta ken dif ferent approaches to address this issue, such as the use of lipid nanoparticles as the delivery vehicle leading to liver accumulation because of its role in first-pass metabolism. "As a consequence the current RNAi approaches most companies utilize are liver specific, even though RNAi can work equally well in non-liver targets if you can deliver the RNA molecules there. Phio's solution to this non-liver delivery challenge was to develop a RNAi platform with structural and chemical modifications that allowes for spontaneous uptake to cells without the need for a delivery vehi- cle," Dispersyn explains. Oisin's PLV delivery technology, meanwhile, has en abled t he compa ny to develop s ys tem ic DNA therapeutics. "Once the first generation of nucleic acid therapeutics have been developed for obvious disease targets, second-generation therapeutics will