Modified Polynucleotides for Protein Expression
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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 low expression rates, especially in primary cells and modified cell lines.
Innovation Solution
Development of nucleosides, nucleotides, and polynucleotides with alternative nucleobases, sugars, or backbones, including specific structures like 5' UTR with Kozak sequences, 3' UTR, 5' cap structures, and poly-A tails, which are codon optimized and purified to enhance intracellular translation.
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 causes alterations and damage
Solution Approach 1:
The patent uses mRNA as an intermediary carrier to transfer genetic information from heterologous DNA to the host cell cytoplasm, bypassing direct genomic integration. The mRNA molecule encodes the desired protein but remains transient in the cytoplasm, allowing protein expression without permanent integration into host DNA, thus avoiding genomic damage while maintaining productivity
Solution Approach 2:
The invention extracts the essential function of gene expression (protein coding information) from the DNA molecule and transfers it to an mRNA carrier. By taking out the coding sequence from heterologous DNA and placing it in an mRNA context with optimized elements (5' cap, 3' poly-A tail, UTRs), the system achieves protein expression without requiring DNA integration into the host genome
2Productivity
If multiple processing steps are used for protein expression (DNA transport to nucleus, transcription to RNA, translation to protein), then protein is produced, but lag times occur before protein generation
Solution Approach 1:
The patent performs preliminary optimization of the mRNA structure before introduction into cells, including adding 5' cap structures, 3' poly-A tails, and optimized UTR regions. These preliminary modifications ensure that once the mRNA enters the cytoplasm, translation can begin immediately without delays for nuclear processing or transcription, significantly reducing lag time
Solution Approach 2:
The invention skips the slow steps of nuclear transport and transcription by introducing pre-synthesized mRNA directly into the cell cytoplasm. This rushes through the expression process by eliminating intermediate steps, allowing immediate translation and protein production, thus reducing overall lag time while maintaining productivity
3Productivity
If DNA is introduced into primary cells or modified cell lines, then protein expression is attempted, but expression rates are low and concentrations are insufficient
Solution Approach 1:
The patent changes critical parameters of the nucleic acid molecule by replacing natural ribose sugars with modified sugars (such as 2'-O-methyl ribose or locked nucleic acid sugars) and incorporating chemically modified bases. These parameter changes in the mRNA structure enhance stability, improve translation efficiency, and increase protein expression rates and concentrations in difficult-to-transfect cells like primary cells and modified cell lines
Solution Approach 2:
The invention creates a composite mRNA structure combining multiple modified elements: modified sugars, modified bases, 5' cap structures, 3' poly-A tails, and optimized UTR regions. This composite approach synergistically enhances translation efficiency and protein expression levels, overcoming the limitations of natural DNA in primary and modified cell lines
Data Source
AI summary
The present disclosure provides alternative sugar moieties and polynucleotides comprising such sugar moieties, and methods of use thereof.