The impact of T7 RNA polymerase class II pause sites on mRNA therapeutics

Background and purpose: There is growing interest in messenger ribonucleic acid (mRNA) drugs for a wide variety of applications including cardiovascular disease. Typically, mRNA drug substances are generated from linear deoxyribonucleic acid (DNA) templates using in vitro transcription (IVT). Thus, the sequence design of the linear DNA templates containing the gene of interest (GOI) plays a critical role in the quality of mRNA therapeutics. A conserved sequence motif ATCTGTT was identified as an endogenous T7 RNA polymerase (RNAP) class II pause site decades ago. However, we do not fully understand the impact of this pause site on the IVT process. This study aimed to characterize T7 RNAP class II pause sites and their impact on mRNA production to optimize therapeutic mRNA design. Methods: Site-directed mutagenesis was performed on DNA templates (e.g., 376, 406, and nuclear green fluorescent protein [nGFP]) to manipulate potential T7 RNAP pause sites. IVT was used to synthesize mRNA, followed by the analysis of transcript integrity using Agilent TapeStation. The effects of mutations on mRNA yield, purity, and immune response (e.g., interleukin-6 [IL-6] secretion in transfected fibroblasts) were evaluated. Results: We found that additional dinucleotides (specifically tc, cc, and ta) downstream of ATCTGTT, or trinucleotides (specifically aaa) immediately downstream of ATCTGT (to give the sequence ATCTGTTtc, ATCTGTTcc, ATCTGTTta, or ATCTGTaaa in the encoded GOI) could cause T7 RNAP termination and produce short abortive transcripts during IVT. The strength of these T7 RNAP pause sites is ATCTGTTtc > ATCTGTaaa > ATCTGTTcc > ATCTGTTta. Furthermore, we find that the location of the T7 RNAP pause site in the GOI also plays a role in the strength of terminating transcription in IVT. The more distant the T7 RNAP pause site from the transcriptional initiation site, the greater the probability of termination. In addition, mRNAs derived from DNA templates harboring T7 RNAP pause sites generated mRNAs with impaired integrity, purity, and mRNA yield. Conclusion: Our study provides insight into the effect of T7 RNAP class II pause sites that may be useful in designing mRNA therapeutics for better integrity, purity, and higher yield.