Synthesis of low immunogenicity RNA with high-temperature in vitro transcription
- Corresponding author: broy{at}neb.com
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.
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Footnotes
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.073858.119.
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Freely available online through the RNA Open Access option.
- Received October 31, 2019.
- Accepted December 30, 2019.
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
