ACE-tRNA Circular DNA for Premature Termination Codon Readthrough
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Solution Overview
Problem
Current therapeutic agents for treating disorders associated with premature termination codons (PTCs) face challenges such as ototoxicity, nephrotoxicity, low readthrough efficiency, and the risk of inserting near-cognate tRNAs that can lead to missense mutations, limiting their clinical effectiveness in addressing genetic diseases like cystic fibrosis.
Innovation Solution
Development of closed-end, circular, non-viral DNA molecules encoding anti-codon edited-tRNAs (ACE-tRNAs) that are free of bacterial sequences and CpG dinucleotides, which can be delivered using minicircles or closed-end DNA threads to specifically revert PTCs back to amino acids, thereby restoring functional protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If aminoglycosides are used as PTC readthrough agents, then readthrough efficiency is improved, but ototoxicity and nephrotoxicity increase
Solution Approach 1:
The patent employs synthetic small-molecule compounds (e.g., tylosin, ataluren) that can be administered as discrete, non-accumulating agents. These compounds bind to the ribosome and facilitate PTC readthrough without the need for continuous exposure to toxic aminoglycosides, thereby maintaining readthrough efficiency while avoiding cumulative ototoxicity and nephrotoxicity.
Solution Approach 2:
The patent modifies the chemical structure and pharmacological properties of PTC readthrough agents by transitioning from natural aminoglycosides to synthetic derivatives. This parameter change in molecular structure allows for reduced toxicity while preserving or enhancing readthrough efficiency, as the synthetic compounds can be optimized for specific biological activities without the harmful side effects of natural aminoglycosides.
2Productivity
If near-cognate tRNAs are inserted to suppress PTCs, then readthrough is achieved, but missense mutations may be generated
Solution Approach 1:
The patent employs small-molecule compounds as intermediary agents that bind to the ribosome and facilitate the insertion of correct amino acids at PTCs. These compounds act as mediators that guide the translation process, ensuring that the correct amino acid is incorporated without causing missense mutations, thereby maintaining both readthrough efficiency and genetic fidelity.
Solution Approach 2:
The patent replaces the natural mechanical process of tRNA recognition and insertion with a chemical mechanism involving small-molecule compounds that bind to the ribosome and induce conformational changes. This chemical substitution allows for more precise control over which amino acid is inserted at the PTC, reducing the risk of missense mutations while maintaining readthrough efficiency.
3Object-affected harmful factors
If Ataluren is used for PTC readthrough, then toxicity is reduced, but readthrough efficiency in human primary cells is low
Solution Approach 1:
The patent divides the PTC suppression mechanism into separate functional components: the small-molecule compound binds to the ribosome, induces conformational changes, and facilitates amino acid insertion. This segmentation of the mechanism allows for optimization of each component independently, enabling improved readthrough efficiency in human primary cells while maintaining low toxicity, as each step can be tuned for specific cellular contexts.
Solution Approach 2:
The patent employs dynamic conformational changes in the ribosome structure induced by small-molecule compounds. These dynamic changes allow the ribosome to transition between different states, facilitating efficient PTC readthrough in human primary cells. The dynamic mechanism enables the compound to adapt to different cellular environments and PTC contexts, improving readthrough efficiency while maintaining low toxicity.
Data Source
AI summary
This invention relates to compositions and methods for treating a disease or disorder associated with premature termination codon. Certain aspects of the invention relate to polynucleotides, vectors, and host cells, and uses thereof.