Removal of dsRNA byproducts using affinity chromatography

Abstract

Double-stranded RNA (dsRNA) molecules are immunogenic byproducts of in vitro transcription of single-stranded RNA (ssRNA). Removal of dsRNA from ssRNA is difficult because the byproducts have similar sizes, sequences, and charges to the desired ssRNA. Here, we describe a dsRNA-specific affinity resin that selectively removes dsRNA from ssRNA. Affinity purification reduced dsRNA levels by >100-fold, to as low as ∼0.00007% w/w of total mRNA, with no negative impact on RNA integrity. The purified RNA, synthesized with standard nucleotides, induced no inflammatory response in a reporter cell line assay designed to measure innate immune responses. Purified RNA induced greater protein expression and healthier cells. The immunogenicity of the affinity-purified RNA with standard nucleotides compares favorably to RNA synthesized with modified nucleotides and purified with cellulose or reverse-phase high-performance liquid chromatography (HPLC). dsRNA affinity purification provides a facile and scalable solution to the problem of immunogenic dsRNA byproducts in transcribed RNA. This approach will improve quality and safety of RNA vaccines and therapeutics.

Keywords: MT: Oligonucleotides: Therapies and Applications; RNA vaccines; circular RNA; double-stranded RNA; in vitro transcription; innate immunity; interferon; messenger RNA; self-amplifying RNA; therapeutic RNA.

Graphical abstract

Figure 1

Comparison of dsRNA affinity resin, cellulose, and RP-HPLC (A) Agarose gel of the GFP mRNA input, or flow-through fraction from blank beads, affinity resin, or cellulose. (B) PAGE of the input dsRNA mixtures, flow-through fraction from blank beads, affinity resin, or cellulose. (C) Immuno-dot blot of samples in B) with dsRNA specific J2 mAb and 100 ng RNA/spot. (D–F) Purification of GFP mRNA with cellulose, RP-HPLC, or affinity resin. (D) J2-immuno dot blot of purified RNAs, with 1 μg RNA/spot. (E) Agarose gel of purified RNAs, visualized with total RNA stain and UV. (F) Dual-color immuno-northern blot with total RNA in red channel, and J2-chemiluminescent signal in green channel. (G) Quantitative dsRNA ELISA analysis of the samples in (D–F). The % dsRNA is plotted on a logarithmic axis, with error bars representing 1 standard deviation. (H) Tabulation of ELISA results.

Figure 2

dsRNA affinity resin column chromatography (A) Overlay of 4 cycles of GFP purification with dsRNA specific affinity resin. The flow through and elution phases are indicated. (B) Agarose gel of the input RNA, 4 flow-through samples, 4 elution samples, and 4 eluates subjected to a heat annealing step. (C) Immuno-northern blot of (B) with total RNA in red channel and J2-chemiluminescent signal in green channel. (D) Quantitative dsRNA ELISA analysis of the purification samples, and % recovery calculated with UV260.

Figure 3

Purification of mRNA produced with standard or optimized IVT (A) Normalized capillary electrophoresis traces of RNA produced with standard or optimized IVT. Input, flow-through, and elution samples are plotted. (B) Agarose gel of samples pre- and post-affinity purification, visualized with automatic exposure and automatic contrast adjustment. (C) Agarose gel with manually enhanced contrast to show minor species. (D) Immuno-northern blot of the samples pre- and post-affinity resin. Due to lower dsRNA levels in the optimized IVT products, a greater mass of RNA was loaded and the contrast was manually enhanced. (E) Plot of all individual ELISA measurements from two assays, run on different days. (F) Quantitative dsRNA ELISA analysis of the standard and optimized replicates, and % integrity measurement calculated from (A).

Figure 4

Transfection of standard and optimized RNA, with and without dsRNA affinity resin purification (A–D) Three wells are transfected with each replicate RNA, and the mean values from all 9 resulting wells are plotted with the error bars representing 1 standard deviation. Non-treated cells (cells) are the negative control and poly-I:C (pIC)-treated cells are the positive control. (A) Interferon expression measured by luciferase reporter present in A549 cells. (B) GFP expression as mean fluorescence intensity measured with a flow cytometer. (C) % cells/debris measured with flow-cytometer. (D) % viability, measured with SYTOX RED viability dye and flow cytometer. ns non-significant, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.