Modified mRNA Nucleosides for Low-Immune Protein Expression
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Solution Overview
Problem
Existing methodologies for protein expression in cells face challenges such as integration of heterologous DNA into host genomic DNA, inefficient processing steps, and immune responses, particularly in primary cells or modified cell lines, leading to altered DNA and reduced expression rates.
Innovation Solution
Development of chemically modified nucleosides, nucleotides, and nucleic acids that reduce innate immune response and enhance intracellular translation efficiency, stability, and protein production, including structures like 5' UTR with Kozak sequences, 3' UTR, 5' cap structures, and poly-A tails, with modifications on nucleobase and sugar portions.
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 modified nucleosides (such as pseudouridine, 5-methylcytidine, and other chemically modified bases) as intermediaries to translate DNA sequences into proteins without requiring integration of heterologous DNA into the host genome. These modified nucleosides are incorporated into mRNA molecules that can be directly translated by cellular machinery, serving as a mediator between genetic information and protein production while avoiding genomic integration
Solution Approach 2:
The invention extracts the essential function of protein expression from the DNA integration process by using mRNA molecules containing modified nucleosides that can be directly translated in the cytoplasm. This separates the protein production function from the nuclear integration step, allowing translation to occur without permanent genomic modification
2Productivity
If multiple processing steps are used for DNA expression (transport to nucleus, transcription, translation), then protein is produced, but lag time increases before protein generation
Solution Approach 1:
The patent performs preliminary action by pre-synthesizing mRNA molecules with modified nucleosides outside the cell (in vitro transcription), then introducing them directly into the cytoplasm. This eliminates the need for nuclear transport and transcription steps that would occur with DNA delivery, allowing immediate translation upon entry
Solution Approach 2:
The invention extracts the time-consuming nuclear transport and transcription steps from the protein expression pathway by using pre-transcribed mRNA molecules. The modified nucleoside-containing mRNA is introduced directly into the cytoplasm where translation occurs immediately, bypassing the nuclear compartment entirely
3Productivity
If DNA is introduced into primary cells or modified cell lines, then expression is achieved, but expression rates are reduced and difficult to obtain
Solution Approach 1:
The patent changes the chemical parameters of the nucleosides by introducing modified bases (pseudouridine, 5-methylcytidine, N1-methyladenosine, and other non-canonical nucleosides) that alter the properties of mRNA. These parameter changes make the mRNA more stable and less immunogenic in primary cells and modified cell lines, significantly improving expression rates and reliability
Solution Approach 2:
The invention applies local quality changes by specifically modifying certain nucleoside positions within the mRNA sequence while leaving other regions unchanged. This allows optimization of specific regions for stability and translation efficiency without affecting the overall coding sequence, enabling tailored mRNA molecules for different cell types
4Object-affected harmful factors
If unmodified nucleic acids are used, then innate immune response occurs, but modified nucleic acids with reduced immune response require complex chemical modifications
Solution Approach 1:
The patent changes the chemical parameters of nucleosides by introducing specific modified bases (pseudouridine at position 5, 5-methylcytidine, N1-methyladenosine, and other modifications) that alter the immunogenicity and stability properties of the mRNA. These parameter changes reduce recognition by innate immune sensors while maintaining translational efficiency
Solution Approach 2:
The invention creates composite nucleic acid structures by combining multiple types of modified nucleosides within a single mRNA molecule. This composite approach uses different modifications at different positions to simultaneously achieve immune evasion, enhanced stability, and optimized translation, with the combined effect greater than individual modifications alone
Data Source
AI summary
The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using them.