Circular dsDNA Composition With Strand-Selective Base Modification
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Solution Overview
Problem
Existing DNA therapeutics face challenges in maintaining stability and reducing immunogenicity while effectively transcribing to produce therapeutic effectors within target cells.
Innovation Solution
Development of chemically modified nucleotides in circular double-stranded DNA (dsDNA) molecules, where one strand contains chemically modified nucleobases and the other is unmodified, along with promoter and effector sequences, to enhance stability and reduce immunogenicity, allowing direct transcription and production of therapeutic proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If chemically modified nucleotides are introduced into dsDNA to reduce immunogenicity, then immunogenicity is reduced, but transcription competence may be compromised
Solution Approach 1:
The patent applies local quality by selectively modifying only the sense strand of the dsDNA with chemically modified nucleotides (such as 5-methylcytidine, 5-hydroxymethylcytidine, or pseudouridine), while leaving the antisense strand unmodified. This localized modification approach reduces immunogenicity in the region where modifications are present, while preserving transcription competence by maintaining the integrity of the complementary antisense strand that is essential for transcription machinery recognition and function.
2Stability of the object's composition
If circular dsDNA is used to enhance stability, then stability is improved, but immunogenicity increases
Solution Approach 1:
The patent applies parameter changes by chemically modifying nucleotides within the circular dsDNA structure. Specifically, it introduces modified nucleotides such as 5-methylcytidine, 5-hydroxymethylcytidine, pseudouridine, or 2'-O-methyl nucleotides into the sense strand. These chemical parameter changes to the nucleotide structure reduce the immunogenicity of the circular DNA while maintaining its enhanced stability, as the circular topology itself is preserved and the modifications are designed to minimize structural disruption.
3Object-affected harmful factors
If one strand is modified while the other remains unmodified, then immunogenicity is reduced, but manufacturing complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the dsDNA into two distinct strands: a sense strand that contains chemically modified nucleotides and an antisense strand that remains unmodified. This segmentation allows for separate synthesis and optimization of each strand, reducing manufacturing complexity compared to modifying the entire DNA structure. The modified sense strand can be synthesized using specialized polymerases and nucleotide pools, while the unmodified antisense strand can be produced by conventional methods, and then annealed together to form the final therapeutic dsDNA.
Data Source
AI summary
The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.