Engineered T7 RNA polymerase to improve mRNA capping efficiency and reduce dsRNA generation during in vitro...

As mRNA emerges as a versatile tool for treating a range of diseases, efficient and scalable production remains an ongoing challenge. Inefficient co-transcriptional incorporation of cap analogs and generation of double-stranded RNA impurities are two of the major challenges that can result in increased manufacturing costs and time, as well as increased immunogenicity and reduced therapeutic potency. In an effort to overcome these challenges, we engineered T7 RNA polymerase variants to enhance capping efficiency and reduce dsRNA generation. In this work, we screened 59 T7 RNA polymerase variants and identified multiple variants, most notably R632N and Q649L, that achieved higher capping efficiency and up to 90% reduction in dsRNA impurity without compromising mRNA integrity. mRNA generated by these engineered T7 RNA polymerase variants showed enhanced translation and reduced immunogenicity in in vitro and in vivo studies. Additionally, they were observed to generate functional, relatively low-immunogenic mRNA without requiring excessive amounts of cap analogs or additional purification steps. Use of such T7 RNAP variants may thus offer a potentially cost-effective strategy for commercial scale mRNA therapeutic manufacturing.