Minimizing dsRNA impurity generation through structure-function-guided directed evolution of T7 RNA polymerase

Area of Science:

• Molecular Biology
• Biotechnology
• RNA Therapeutics

Background:

• Double-stranded RNA (dsRNA) is a pathogen-associated molecular pattern that activates innate immunity, posing challenges for mRNA therapeutics.
• Current strategies for dsRNA reduction in mRNA synthesis often involve T7 RNA polymerase (T7 RNAP) engineering.

Purpose of the Study:

• To elucidate the mechanisms of dsRNA generation during T7 RNAP-catalyzed in vitro transcription.
• To engineer T7 RNAP variants with significantly reduced dsRNA byproduct formation for improved mRNA therapeutics.

Main Methods:

• Investigated the role of the RNA exit tunnel in transcription termination and dsRNA formation.
• Employed phage-assisted non-continuous evolution to engineer T7 RNAP variants.
• Introduced specific mutations within the RNA exit tunnel of T7 RNAP.

Main Results:

• The RNA exit tunnel is critical for both transcription termination and dsRNA byproduct generation.
• Engineered T7 RNAP variants with mutations in the RNA exit tunnel showed reduced dsRNA.
• A double mutant T7 RNAP (M183E + I210V) eliminated over 99% of dsRNA.

Conclusions:

• The RNA exit tunnel of T7 RNAP is a key determinant of dsRNA byproduct formation.
• Engineered T7 RNAP variants, particularly the M183E + I210V double mutant, significantly minimize dsRNA content.
• These engineered polymerases are highly promising for developing next-generation mRNA therapeutics with enhanced safety and efficacy.