Double-stranded RNA reduction by chaotropic agents during in vitro transcription of messenger RNA

In-vitro-transcribed messenger RNA (mRNA) has recently shown increasing significance in the development of vaccines and therapeutics. Immunogenic double-stranded RNA (dsRNA) is an undesired byproduct formed during in vitro transcription (IVT), and it is challenging to reduce dsRNA byproduct from mRNA due to their similar sizes and intrinsic characteristics. Removal of dsRNA relies heavily on post-IVT chromatography purifications, such as reverse-phase high-pressure liquid chromatography, which increase manufacturing costs, reduce yield, and often decrease integrity, especially for long mRNA. Thus, it would be ideal to reduce and control the level of dsRNA during IVT. We herein present a simple, scalable, and controllable method to reduce the formation of dsRNA byproducts during IVT. Selected chaotropic agents at optimized concentrations are included during IVT to create a mild denaturing environment to prevent the undesired intermolecular or intramolecular base-pairing that is thought to promote RNA-templated dsRNA formation by RNA polymerase. Compared with regular IVT, our improved method produces mRNA with significantly less dsRNA, much lower immuno-stimulation, and more efficient protein expression. Therefore, this method potentially eliminates dsRNA removal purification steps and does not require reduced magnesium concentration, elevated temperature, or custom reagents, enabling a straightforward, high-yield, and cost-effective scale-up approach for mRNA manufacturing. [Display omitted] Addition of chaotropic agents to in vitro transcription reactions minimizes the formation of undesired double-stranded RNA byproducts while maintaining yield and transcript integrity, obviating the need for harsh or time-consuming HPLC purification techniques.