Abstract
The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications.
Highlights
In recent years, in vitro-transcribed (IVT) mRNA for research and therapeutic applications has gained tremendous interest
DsRNA Contaminants in IVT mRNAs Bind to Cellulose in the Presence of Ethanol To test the feasibility of applying cellulose to remove double-stranded RNA (dsRNA) contaminants from IVT RNA, first a simple separation experiment was performed (Figure S1)
The dot blot analysis performed with J2 dsRNA-specific antibody demonstrated that the fraction of unbound mRNA contained approximately 90% less dsRNA compared to the corresponding input mRNA, confirming the efficient removal of dsRNA contaminants by cellulose (Figure 1A)
Summary
In vitro-transcribed (IVT) mRNA for research and therapeutic applications has gained tremendous interest (reviewed by Sahin et al.[1]). The simplicity of the approach to synthesize almost any protein in vitro or in vivo by delivering the encoding IVT mRNA makes it very appealing. A promoter-independent transcription of full-length anti-sense RNA has been reported as a novel mechanism of dsRNA generation in T7RNAPol-driven IVT reaction.[13]
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