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Science in Lab | Investigating the Influence of dsRNA Byproducts on IVT mRNA Immunogenicity

mRNA vaccines, a rule-changing breakthrough. The COVID-19 pandemic has made the nucleic acid drug mRNA technology a hit.

mRNA technology can theoretically produce any kind of protein needed by humans, whether it is in the field of prevention or in the treatment of diseases, and its value is self-evident. In addition to the technical efficacy of the attack, safety, tolerance and immunogenicity are also mRNA therapy can not get around the topic.

Dr. Şahin, CEO of BioNTech, suggests that addressing the immunogenicity triggered by mRNAs and their vectors is one of the biggest challenges that need to be addressed in the development of mRNA vaccines and therapies. Exogenous mRNAs are immunostimulatory in their own right and are recognized by a wide range of cell surface, endosomal and cytoplasmic innate immune receptors; while another major trigger for immune response comes from contaminants present in the IVT response, with double-stranded RNAs (dsRNAs) being the major by-products of the IVT response.

Mechanism of dsRNA formation in IVT

The production of dsRNA byproducts occurs mainly through two different mechanisms. First, based on the RdRp activity of T7RNA polymerase itself, it is able to form a hairpin structure by using the product RNA as a template and continuing to extend the RNA at the 3' end after reverse complementary pairing with the original RNA molecule (Figure.1a); alternatively, the randomly broken/incomplete transcriptional product is complementarily paired with the mRNA during transcription and thus extended to form an incomplete double-stranded RNA (Figure.1b), and secondly, it is also possible that dsRNA is formed through a promoter-independent transcription process using the DNA antisense strand as a template (Figure. 1c).

Figure. 1 Schematic representation of the possible mechanisms of dsRNA byproduct formation during transcription in vitro

dsRNA causes immunogenicity

RIG-I and MDA5 are two major cytoplasmic sensors that, upon recognition of viral dsRNAs, activate the antiviral signaling pathway, leading to transcriptional up-regulation of type I and type III interferons (ifn).RIG-I and MDA5 have different RNA specificities as a way of recognizing various types of viruses.RIG-I primarily recognizes the termini of the dsRNAs, especially the 5' triple phosphate group (5'ppp), whereas MDA5 recognizes long dsRNAs (0.5-1kb) in a manner that depends on the double-strand length. More detailed structural and biochemical analyses indicate that MDA5 forms filaments along the length of the dsRNA, and that filament formation is critical for antiviral signaling activation and dsRNA length detection. mDA5 also hydrolyzes ATP only upon binding to the dsRNA, although ATP hydrolysis regulates the activity of MDA5 in seemingly complex ways. It has long been assumed that the immunostimulatory activity of T7pol transcripts is due to the fact that they contain 5'ppp, which stimulates RIG-I. However, the data suggest that removal of 5'ppp by phosphatase does not completely suppress immunogenicity. It was demonstrated that T7pol frequently produces high levels of unintended double-stranded RNAs (dsRNAs) that are highly immunostimulatory and consist of the intended sense transcript and its fully complementary antisense transcript.



Figure 2 dsRNA activates antiviral natural immune signaling pathway through intracellular receptors

Detection and regulation of dsRNA

In April 2023, the United States Pharmacopeia updated the "Methods for Quality Analysis of mRNA Vaccines-Draft Guidance", which added the ELISA method for detecting dsRNA residues. The current methods for detecting dsRNA residues in mRNA vaccines contain the classical Western blotting and the ELISA. Western blotting is performed by spot sampling dsRNA standards and RNA samples to be tested onto a positively charged nylon membrane for adsorption and fixation, incubating the membrane with a primary antibody capable of specifically recognizing double-stranded RNA, and then using enhanced chemiluminescence (ECL) reagents to develop the color after addition of an HRP-labeled secondary antibody for incubation. The concentration of dsRNA in the sample is analyzed by comparing the fluorescence signal of the sample with a dsRNA standard of known concentration. dsRNA detection by ELISA is generally based on a double antibody sandwich enzyme immunoassay using a capture antibody-coated microtiter plate to form a solid-phase antibody, to which a dsRNA calibrator and the sample to be tested are added, followed by the addition of the detection antibody, and finally by the addition of Then the detection antibody is added, and finally the horseradish peroxidase (HRP)-labeled ELISA secondary antibody is added to form a complex of "Coated Antibody - dsRNA - ELISA Detection Antibody", which is washed and then added to the color development solution to develop the color, and then the reaction is terminated by using an ELISA to detect the absorbance value, which is correlated with the amount of dsRNA in the samples. Vazyme EasyAna dsRNA (Modified) Quantitative Detection Kit (ELISA) 2.0- DD3509EN applys double-antibody sandwich ELISA to detect the residual double-stranded RNA (dsRNA). It’s easy to use and can be accurately quantified,and now is available >>

For the inhibition of dsRNA byproducts, the main approaches have been related to the use of modified nucleotides, reduction of MgCl2 concentration, alteration of template sequences without promoter termini, or purification. Modified nucleotides reduce dsRNA production as a byproduct of the IVT reaction, which is at least partially responsible for the diminished immunogenicity of T7 transcripts caused by modified nucleotides. At the same time, the insertion of the modified nucleotide had no effect on the immunogenicity of the dsRNA itself. When the concentration of magnesium ions was as low as a certain concentration, dsRNA production was barely detectable. In a previous study, it was shown that contaminating dsRNA could be efficiently and effectively removed from IVT mRNA by chromatographic methods such as reversed-phase fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC).Strikingly, the purification of FPLC has been shown to increase the protein yield of IVT mRNA by 1,000-fold in primary human dendritic cells. Thus, proper purification of IVT mRNA appears to be essential for maximizing protein (immunogen) production in dendritic cells and avoiding unwanted innate immune activation.


Figure 3 Effect of nucleoside modification and FPLC purification on innate immunity perception


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[1] Pardi, N. , et al. "mRNA vaccines — a new era in vaccinology." Nature Reviews Drug Discovery (2018).

[2] Wu, M. Z. , et al. "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription." (2019).

[3] Yoneyama, M. , and  T. Fujita . "Recognition of viral nucleic acids in innate immunity." Reviews in Medical Virology 20.1(2010):4-22.