Modified mRNA with Reduced Uracil Content
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Solution Overview
Problem
Current methods for protein expression in cells face challenges such as integration of heterologous DNA into host genomic DNA, leading to alterations, and require multiple processing steps, resulting in lag times and difficulties in achieving reasonable expression rates or concentrations, especially in primary cells or modified cell lines. Additionally, DNA expression often induces an innate immune response.
Innovation Solution
Development of mRNA molecules with altered uracil content, specifically incorporating 5-methoxy-uracil, which reduces the innate immune response and enhances protein expression by optimizing nucleic acid-based therapeutics, improving expression rates and stability, and minimizing bio-responses like immune activation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If heterologous DNA is introduced into cells for protein expression, then protein production is achieved, but integration into host genomic DNA occurs causing alterations and damage
Solution Approach 1:
The patent extracts the problematic DNA intermediate step by directly introducing mRNA into cells, bypassing the need for heterologous DNA integration into the genome. This eliminates the harmful genomic alterations while maintaining protein production capability through direct translation of the introduced mRNA
Solution Approach 2:
The patent uses mRNA as an intermediary carrier that temporarily delivers the protein-coding information to the cell without requiring permanent genomic integration. The mRNA acts as a transient mediator that enables protein expression without the harmful effects of DNA integration
2Productivity
If multiple processing steps are used for protein expression from DNA, then protein is produced, but lag times occur before protein generation
Solution Approach 1:
The patent performs preliminary action by pre-synthesizing the mRNA in vitro before introducing it to the target cells. This eliminates the time required for transcription in vivo and allows immediate translation upon introduction, reducing overall lag time
Solution Approach 2:
The patent extracts and eliminates the transcription step from the cellular process by providing pre-transcribed mRNA. This removes one of the multiple processing steps that cause lag time, allowing direct translation of the introduced mRNA into protein
3Productivity
If standard mRNA with normal uracil content is used, then protein expression is achieved, but innate immune response is activated
Solution Approach 1:
The patent changes the chemical parameter of uracil content in mRNA by replacing a portion of uracil nucleotides with alternative nucleotides (such as 5-methyluracil or pseudouracil). This chemical modification reduces recognition by innate immune sensors while maintaining translation efficiency, thereby suppressing immune activation
Solution Approach 2:
The patent converts the potentially harmful high uracil content that triggers immune response into a benefit by strategically replacing specific uracils with alternative nucleotides. This modification reduces immunogenicity while preserving or enhancing protein expression, turning the immune activation problem into improved therapeutic efficacy
Data Source
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AI summary
The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.