Cold-Active RNA Polymerases for Low-dsRNA In Vitro Transcription
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Solution Overview
Problem
Existing RNA polymerases, such as T7 RNA polymerase, produce immunostimulatory byproducts like degraded or uncapped RNA molecules and double-stranded RNA (dsRNA) during in vitro transcription, leading to off-target immune stimulation, which is a barrier to the development of mRNA drugs beyond immunotherapies.
Innovation Solution
Utilization of cold-active RNA polymerases, derived from bacteriophages like Pseudomonas phage Njord, which operate at lower temperatures and produce transcripts with reduced immunogenicity and fewer byproducts, such as dsRNA, by minimizing self-annealing and promoting capped RNA synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If T7 RNA polymerase is used for in vitro transcription, then high transcription efficiency is achieved, but immunostimulatory byproducts such as degraded RNA, uncapped RNA, and double-stranded RNA are generated
Solution Approach 1:
The patent changes the temperature parameter from standard physiological temperature to lower temperatures (0-30°C), which fundamentally alters the transcription kinetics and product profile. This parameter change reduces the formation of immunostimulatory byproducts while maintaining transcription efficiency, directly resolving the contradiction between productivity and harmful factor generation
Solution Approach 2:
The patent converts the previously harmful low-temperature condition (which was thought to reduce enzyme activity) into a beneficial feature. By discovering that cold-active RNA polymerases thrive at low temperatures and produce fewer immunostimulatory byproducts, the patent transforms a limitation into an advantage, eliminating harmful factors while maintaining or improving transcription efficiency
2Quantity of substance
If standard in vitro transcription is performed, then RNA production is achieved, but purification requirements are high due to byproduct contamination
Solution Approach 1:
The patent converts the previously harmful low-temperature condition into a beneficial feature that inherently reduces byproduct formation. Cold-active RNA polymerases produce cleaner transcripts with fewer immunostimulatory contaminants, thereby reducing purification requirements while maintaining high RNA production levels
Solution Approach 2:
By changing the temperature parameter to low temperatures and using cold-active RNA polymerases, the patent alters the transcription reaction conditions to inherently produce fewer contaminants. This parameter change reduces the burden on downstream purification processes while maintaining high RNA yield
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The use of cold-active RNA polymerases results in higher quality mRNA drug substances with reduced immunogenicity and lower purification requirements, enabling the broader application of mRNA therapies.
Implementation Method 1
cold-active RNA polymerases, derived from bacteriophages like Pseudomonas phage Njord, which operate at lower temperatures and produce transcripts with reduced immunogenicity
Data Source
AI summary
The present disclosure relates, according to some embodiments, to cold-active RNA polymerases, variants thereof, compositions and kits comprising cold-active RNA polymerases, and methods of using cold-active RNA polymerases. Cold-active polymerases may have, for example, an amino acid sequence ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% identical (e.g., ≥95% or ≥98% identical) to any of SEQ ID NOS: 1-19 and optionally may have at least one substitution relative to SEQ ID NO:1.