Region-Specific DNA Methylation for Gene Expression Control
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Solution Overview
Problem
Existing DNA molecules used in gene therapies lack control over methylation, leading to immunostimulatory and toxic effects on eukaryotic cells and silencing of gene expression due to unmethylated CpG motifs.
Innovation Solution
The production of DNA products with controlled unmethylated and methylated regions by appending specific DNA molecules to create DNA products with altered immunomodulatory effects, reducing immunostimulation and enhancing gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If unmethylated DNA is used in gene therapy, then gene expression can be achieved, but immunostimulatory and toxic effects occur due to unmethylated CpG motifs
Solution Approach 1:
The patent applies local quality by differentiating methylation status across different regions of the DNA molecule. The promoter region is kept unmethylated to ensure gene expression, while the coding sequence region is methylated to reduce immunostimulatory effects. This spatial differentiation of methylation properties resolves the contradiction between achieving gene expression and minimizing immune activation.
Solution Approach 2:
The DNA molecule is segmented into distinct functional regions with different methylation characteristics. The promoter sequence (unmethylated) and coding sequence (methylated) are treated as separate segments with different immunomodulatory properties. This segmentation allows each region to fulfill its specific function while collectively reducing overall immunotoxicity.
2Object-affected harmful factors
If DNA methylation is increased to reduce immunostimulation, then immunomodulatory effects are reduced, but promoter regions may be methylated leading to gene silencing
Solution Approach 1:
The invention applies local quality by assigning different methylation states to different functional regions of the DNA. The promoter region is specifically protected from methylation to maintain transcriptional activity, while the coding sequence is methylated to reduce immune recognition. This localized control prevents the trade-off between immunosuppression and gene silencing.
Solution Approach 2:
The patent employs preliminary action by pre-methylating the DNA at specific positions before introducing it into eukaryotic cells. Methyltransferase enzymes are used to establish the desired methylation pattern in advance, ensuring that the promoter remains unmethylated while the coding sequence is methylated, thereby preventing subsequent gene silencing while reducing immunostimulation.
3Manufacturing precision
If methyltransferase enzymes are used to methylate DNA, then methylation control is improved, but lack of precise control results in promoter region methylation and gene silencing
Solution Approach 1:
The patent achieves local quality by designing the DNA construct with specific methylation sites positioned only in the coding sequence region, away from the promoter. The methyltransferase enzyme is guided to these specific positions through the DNA sequence design, ensuring methylation occurs only where desired and never at the promoter, thus maintaining gene expression while achieving precise methylation control.
Solution Approach 2:
The invention uses the DNA sequence structure itself as an intermediary to guide the methyltransferase enzyme. Specific sequence features in the coding region serve as recognition sites for the methyltransferase, directing methylation activity to the correct location while the promoter region's sequence structure prevents enzyme binding, thereby mediating precise spatial control of methylation.
Data Source
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AI summary
The invention relates to DNA molecules with a methylated region and an unmethylated region (e.g. with immunomodulatory properties), and uses thereof. The invention also relates to methods for producing DNA molecules and kits for use in such methods.