Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Abstract

transcribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-extension of the run-off product. We show that template-encoded poly(A) tailing does not affect 3'-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.