Engineered chimeric T7 RNA polymerase improves salt tolerance and reduces dsRNA impurity generation during <i>in vitro</i> transcription of mRNA

Messenger RNA (mRNA) is typically produced enzymatically through in vitro transcription using wild-type T7 RNA polymerase; however, this enzyme is also known to generate double-stranded RNA (dsRNA) impurities during transcription. These impurities may evoke an immune response, potentially reducing the therapeutic index of the drug product. Upstream and downstream approaches may be used to mitigate the formation or removal of such dsRNA impurities. However, these processes can be costly, reduce yield, and be challenging to scale for clinical manufacturing. In this work, we engineered a chimeric T7 RNA polymerase by tethering a DNA-binding domain to increase the selectivity of the polymerase for DNA templates and found that it was capable of reducing dsRNA formation. The chimeric T7 RNA polymerase reduced dsRNA levels by three- to four-fold relative to wild-type T7 RNA polymerase with commensurate reduction in immune stimulation in vitro. Additionally, the chimeric T7 RNA polymerase showed improved salt tolerance and was active at NaCl concentrations up to 150 mM, which is otherwise a restrictive condition for wild-type T7 polymerase. These features make this novel enzyme an attractive option for addressing various challenges facing the field of mRNA manufacturing.