An origin of the immunogenicity of in vitro transcribed RNA

Abstract

The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg2+ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated.

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

“…We wanted to investigate whether high-temperature IVT affected the formation of antisense-dependent dsRNA structures. Because we were unable to detect the formation of the antisense RNA in any of our templates under native conditions (data not shown), we tested the same sequence that was reported in the previous study (template 512B) (Mu et al 2018). With the 512B template, we were able to detect the formation of RNA molecules that were sensitive to RNase III treatment when transcription was performed with either wild-type RNAP (Fig.…”

“…A previous report has demonstrated the synthesis of antisense RNA molecules that can base-pair with the run-off transcript and contribute to the formation of dsRNA contaminants in the IVT reaction (Mu et al 2018). We wanted to investigate whether high-temperature IVT affected the formation of antisense-dependent dsRNA structures.…”

“…The second type of dsRNA molecules is formed by the hybridization of an antisense RNA molecule to the run-off transcript. The antisense RNA molecules have been reported to be formed in a promoter-and run-off transcript-independent manner (Mu et al 2018). The mechanistic and structural requirements for the formation of these by-products are not well understood, and therefore, it is a challenge to devise methods to prevent their formation or enable their removal.…”

mRNA as a Transformative Technology for Vaccine Development to Control Infectious Diseases

“…A highly purified RNA drug substance is critical for the potency of an mRNA vaccine, because contaminants can activate non-specific or undesirable innate sensors. [34][35][36][37] Karikó et al, 36,38 and more recently Foster et al, 39 have demonstrated that purification of mRNA from double-stranded RNA (dsRNA) contaminants can enhance in vivo translation and reduce innate immune activation, and is critical for the success of RNA-based gene therapy and CAR T cell therapy applications. After purification, mRNA is exchanged into a final storage buffer or formulated with the delivery system for use.…”

Double-Stranded RNA Sensors and Modulators in Innate Immunity

“…In vitro transcription with T7 phage RNA polymerase is one of the most common methods for preparing RNAs. But it is known to generate a variety of unintended by-products, one of which is dsRNA formed by intended sense RNA and fully complementary antisense RNA (9). This can lead to misdefining RNA specificity for a protein that is in fact dsRNA specific (10).…”