Chemically Altered mRNA for Protein Expression
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Solution Overview
Problem
Current methodologies 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 low expression rates, especially in primary cells or modified cell lines.
Innovation Solution
Development of mRNA molecules with chemical alterations, such as alternative uracil and cytosine, which are incorporated in specific percentages to enhance therapeutic and diagnostic applications by improving intracellular translation efficiency and reducing innate immune response.
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 uses mRNA as an intermediary carrier to transfer genetic information from DNA to protein without requiring DNA integration into the host genome. The mRNA molecules are introduced directly into cells, where they are translated by cellular machinery to produce the desired protein, thus achieving protein expression while avoiding genomic DNA damage
Solution Approach 2:
The patent extracts the essential function of genetic information transfer by using synthesized mRNA molecules that contain only the necessary coding sequence for the desired protein. This eliminates the need for introducing entire plasmids or genomic DNA, thereby preventing integration events and genomic alterations while maintaining protein production capability
2Productivity
If DNA is introduced into cells for protein expression, then protein production is possible, but multiple processing steps are required resulting in lag times
Solution Approach 1:
The patent performs preliminary action by synthesizing the mRNA molecules with all necessary processing features already incorporated before introduction into cells. The mRNA is pre-formed with appropriate 5' caps, 3' poly-A tails, and coding sequences optimized for translation, eliminating the need for cellular transcription and processing steps that would otherwise be required for DNA-based expression
Solution Approach 2:
The patent extracts and eliminates the time-consuming intermediate steps of DNA transcription and RNA processing by directly introducing the final translation-ready mRNA product into cells. This bypasses the multi-step DNA expression pathway (DNA import → nuclear transport → transcription → RNA processing → cytoplasmic translation) and goes directly to the translation step
3Productivity
If DNA is introduced into primary cells or modified cell lines, then protein expression is attempted, but expression rates are low or expression does not occur
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of nucleotides in the mRNA molecules. Specific percentages of uracil and cytosine bases are replaced with alternative nucleotides (such as 5-methylcytosine, pseudouridine, or other modified bases) to optimize the mRNA's stability, translation efficiency, and immune evasion properties in primary cells and modified cell lines
Solution Approach 2:
The patent applies local quality by making specific modifications at particular positions within the mRNA sequence. Different nucleotide modifications are applied at different locations and in different proportions throughout the mRNA molecule, creating locally optimized regions that enhance translation efficiency while maintaining overall gene expression fidelity
Data Source
AI summary
The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them.