mRNA In Vitro Transcription Template with 5' Non-Coding Sequence
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Solution Overview
Problem
Existing methods for in vitro transcription of mRNA are challenged by the presence of double-stranded RNA (dsRNA) contaminants, which stimulate immune responses and are difficult to remove, leading to inefficient and costly mRNA production for therapeutic applications.
Innovation Solution
The method involves synthesizing a DNA template with a 5′-non-coding sequence (NCS) upstream of the phage DNA-Dependent RNA Polymerase promoter, followed by PCR amplification and in vitro transcription using a phage DNA-dependent RNA polymerase transcription system, resulting in an RNA composition with reduced dsRNA levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional in vitro transcription methods are used, then mRNA production is achieved, but dsRNA contaminants are generated that stimulate immune responses
Solution Approach 1:
The patent applies preliminary action by incorporating a 5′ non-coding sequence (NCS) of at least 150 nucleotides upstream of the T7 promoter in the DNA template design. This pre-designed feature prevents dsRNA formation at the source during transcription initiation, eliminating the need for post-transcriptional purification steps to remove immunogenic contaminants.
Solution Approach 2:
The invention extracts and removes the problematic region by designing the NCS to extend upstream beyond the typical promoter region. This extended sequence acts as a buffer that prevents the formation of dsRNA structures that would otherwise be generated during transcription, effectively taking out the source of contamination before it can affect the final mRNA product.
2Object-affected harmful factors
If dsRNA removal methods are applied, then immune response is reduced, but production time and cost increase
Solution Approach 1:
The patent converts the potential harm of dsRNA formation into a benefit by designing the NCS to naturally prevent dsRNA generation during transcription. The extended 5′ non-coding sequence serves as a protective element that transforms the transcription process itself into a dsRNA-free method, eliminating the need for time-consuming removal steps while maintaining high mRNA yields.
3Object-affected harmful factors
If dsRNA removal methods are applied, then immune response is reduced, but production cost increases
Solution Approach 1:
The invention implements self-service by incorporating the dsRNA prevention feature directly into the DNA template design. The 5′ non-coding sequence of at least 150 nucleotides automatically prevents dsRNA formation during the transcription process itself, making the system self-correcting and eliminating the need for additional purification reagents, steps, or specialized equipment that would increase manufacturing costs.
4Object-generated harmful factors
If template modifications are made to reduce dsRNA, then dsRNA levels decrease, but RNA yield may be affected
Solution Approach 1:
The patent applies parameter changes by modifying the length and position of the 5′ non-coding sequence in the DNA template. By extending this sequence to at least 150 nucleotides upstream of the T7 promoter, the transcription parameters are optimized to prevent dsRNA formation while maintaining efficient transcription initiation and elongation, thereby preserving high RNA yields without compromising purity.
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
This approach significantly reduces the levels of dsRNA in the RNA composition, thereby minimizing immune responses and improving the efficiency and safety of mRNA production for therapeutic uses, while maintaining RNA yields comparable to traditional methods.
Implementation Method 1
transcribing the IVT-template in vitro using a phage DNA-Dependent RNA Polymerase transcription system to prepare an RNA composition
Implementation Method 2
amplifying the DNA-template using a polymerase chain reaction (PCR) to produce an in vitro transcription template (IVT-template)
Data Source
AI summary
The present application provides improved methods and compositions for in vitro transcription of mRNA that has reduced levels of dsRNA contaminant.