Purified mRNA with Modified Nucleosides Reducing Immunogenicity
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Solution Overview
Problem
Current RNA delivery methods face challenges due to the immunogenicity and contamination issues associated with RNA transcribed by phage RNA polymerases, which retain low levels of activation of innate immune pathways despite nucleoside modifications, and contain aberrant RNAs and dsRNA contaminants.
Innovation Solution
The development of purified mRNA preparations enriched with modified nucleosides such as 1-methyl-pseudouridine and m5C, which undergo enzyme digestion and chromatography processes to reduce immunogenicity and enhance translation efficiency, are encapsulated in lipid nanoparticles for delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RNA is transcribed by phage RNA polymerases with modified nucleosides, then translation efficiency is improved, but immunogenicity is not sufficiently reduced due to remaining activation of innate immune pathways
Solution Approach 1:
The patent extracts and removes dsRNA contaminants and aberrant RNAs from the transcription mixture through purification steps including DNase treatment, phenol-chloroform extraction, and ethanol precipitation. This separation isolates the desired modified mRNA from harmful contaminants that activate innate immune pathways, thereby reducing immunogenicity while preserving the translation efficiency provided by the modified nucleosides.
Solution Approach 2:
The patent changes the chemical composition parameters by incorporating specific modified nucleosides (1-methyl-pseudouridine and m5C) at controlled ratios during transcription. These parameter changes in nucleoside modification suppress recognition by innate immune sensors while maintaining high translation efficiency, resolving the contradiction between reduced immunogenicity and improved productivity.
2Manufacturing precision
If purified mRNA preparations are produced through enzyme digestion and chromatography, then purity is improved, but manufacturing complexity increases
Solution Approach 1:
The patent segments the purification process into distinct sequential steps: (1) DNase treatment to remove DNA contaminants, (2) Phenol-chloroform extraction to separate RNA from proteins and other contaminants, (3) Ethanol precipitation to isolate pure RNA, and (4) Resuspension in sterile buffer. This segmentation makes the complex purification process more manageable and reproducible while achieving high purity mRNA preparations.
Solution Approach 2:
The patent uses intermediary substances during purification: phenol-chloroform as a liquid-liquid extraction mediator to separate RNA from contaminants, ethanol as a precipitation mediator to isolate pure RNA, and RNase-free water or buffer as a resuspension mediator. These intermediaries facilitate each purification step while maintaining RNA integrity, balancing purity achievement with process feasibility.
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 purified mRNA preparations demonstrate significantly reduced immunogenicity and enhanced translation efficiency when delivered to mammalian cells, effectively minimizing activation of innate immune pathways and improving protein expression.
Implementation Method 1
subjecting the preparation of messenger RNA comprising at least one modified nucleoside to at least one purification process selected from the group consisting of enzyme digestion and chromatography
Implementation Method 2
subjecting the preparation of messenger RNA comprising at least one modified nucleoside to at least one purification process selected from the group consisting of enzyme digestion and chromatography
Data Source
AI summary
This invention provides purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising a modified nucleoside, and methods of assessing purity of purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising a modified nucleoside.