Self-amplifying RNA (saRNA) manufacturing places stringent demands on in vitro transcription (IVT), particularly for long templates where polymerase processivity, transcript integrity, capping efficiency, yield, and double-stranded RNA (dsRNA) formation directly influence product quality and downstream purification burden. This talk describes an independent third-party evaluation conducted by Hongene Biotech comparing Prima RNApols® ExTend Cap AU with wild-type T7 RNA polymerase for transcription of a 9.5 kb linearized saRNA template encoding a Firefly luciferase reporter. IVT reactions were performed under matched conditions using 9 mM NTPs, 4 mM Cap AU, 37 °C incubation, and a 2-hour reaction time. Product quality attributes were assessed by immunoassay-based dsRNA quantification, LC-MS capping analysis, fragment analyzer integrity assessment, and UV-based yield measurement.
The results demonstrate that polymerase selection substantially affects saRNA IVT outcomes. Prima ExTend Cap AU produced approximately an order of magnitude less dsRNA than wild-type T7 while maintaining comparable capping efficiency, with 98% capping for ExTend Cap AU versus 99% for T7. Transcript integrity was approximately 11% higher with ExTend Cap AU, indicating fewer short or truncated RNA species and a cleaner IVT product profile. Total yield remained within 10% of T7, at 8.2 mg/mL for ExTend Cap AU compared with 9.2 mg/mL for T7. Because ExTend Cap AU combined low dsRNA, high capping efficiency, improved integrity, and near-equivalent yield, the talk highlights its potential to increase effective target RNA yield and reduce downstream processing burden in saRNA manufacturing.
Overall, the presentation emphasizes that engineered RNA polymerases can improve critical quality attributes without imposing major trade-offs in yield or capping, supporting more efficient and scalable production of high-quality saRNA.
Malithi Jayasinghe
Malithi Jayasinghe, PhD is a Scientist at Primrose Bio Inc. specializing in RNA polymerase engineering for RNA therapeutics, including mRNA vaccines and self-amplifying RNA applications. Her work applies ultra-high-throughput screening and enzyme optimization to improve IVT performance, with a focus on reducing dsRNA byproducts, enhancing capping efficiency, and increasing mRNA yield. She has also contributed to the development of novel enzymatic oligonucleotide synthesis methods designed to support more robust and cost-effective therapeutic oligonucleotide production. Malithi earned her PhD from The Ohio State University, where she studied a unique RNA polymerase family and its biotechnological applications. Her expertise spans RNA biochemistry, enzymology, molecular biology, RNA–protein interactions, and enzyme kinetics.