Abstract
The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-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.
Highlights
One prominent use of in vitro transcription (IVT) in the past few years has been to generate mRNAs for biopharmaceutical and therapeutic applications
We describe the use of thermostable T7 RNA polymerases (RNAPs) and the effect of the temperature of the IVT reaction in the formation of double-stranded RNA (dsRNA) by-products
We focused on commercially available thermostable (Ts) RNAPs (TsT7-1 and TsT7-2; Supplemental Fig. S4), which allowed us to raise the temperature of the IVT reaction
Summary
One prominent use of in vitro transcription (IVT) in the past few years has been to generate mRNAs for biopharmaceutical and therapeutic applications. It is known that introduction of synthetic in vitro transcribed mRNAs into cells or animal models results in an immune response against the synthetic molecules (Weissman et al 2000; Karikó et al 2004; Akira et al 2006; Sahin et al 2014; Freund et al 2019). Such outcomes are undesirable in therapeutic applications in which an immune response is detrimental or unnecessary (for example, protein-replacement therapies).
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