Improvement Of Self-Amplifying RNA Quality And Potency With IVT Optimization

By J. Salnikov, R. Bushko, Y. Kong, J. Posy

Working with large and self-amplifying RNAs (saRNA) presents unique challenges that require careful optimization of various factors to ensure high yields and quality. In this study, we focused on enhancing the production of saRNA by refining the conditions of the In-Vitro Transcription (IVT) reaction. Many commercially available IVT buffers are not tailored specifically for saRNA, which can lead to suboptimal results.

To address this, we systematically evaluated several key parameters, including incubation temperatures, magnesium source and concentration, and reaction buffer compositions. By fine-tuning these conditions, we were able to significantly improve the synthesis process, enabling us to successfully produce large saRNA constructs up to 16kb in length. These optimizations led to higher yields, minimal residual double-stranded RNA (dsRNA), and improved potency of the final RNA product.

This work represents a crucial step in optimizing saRNA production for therapeutic applications, ensuring more efficient and reliable RNA-based therapies.