TREMs for Premature Termination Codon Read-Through
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods fail to effectively modulate protein expression in cells with premature termination codons (PTCs), leading to truncated proteins and associated diseases, as they cannot efficiently read through or correct these codons.
Innovation Solution
Modified tRNA-based effector molecules (TREMs) are used to pair with premature termination codons, allowing for the incorporation of amino acids and restoration of full-length protein expression by contacting cells with TREM compositions that include specific anticodons and modifications such as 2′-O-MOE, pseudouridine, or 5,6 dihydrouridine.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used to treat cells with premature termination codons, then the treatment is simple, but the methods fail to effectively modulate protein expression and produce truncated proteins
Solution Approach 1:
The patent employs composite tRNA structures (TREM compositions) that integrate multiple functional domains including modified nucleotides (2′-O-MOE, pseudouridine, 5,6 dihydrouridine), specific anticodon sequences targeting PTCs, and engineered amino acid acceptance capabilities. This composite design enables simultaneous PTC recognition and functional readthrough, resolving the contradiction between treatment effectiveness and structural simplicity.
Solution Approach 2:
The invention modifies key parameters of tRNA molecules including nucleotide composition (incorporating rare or modified bases), anticodon sequence (matched to specific PTCs like UAA, UAG, UGA), and amino acid charging properties. These parameter changes transform conventional tRNA into specialized TREM compositions capable of effective PTC modulation while maintaining manageable complexity through systematic modification.
2Productivity
If TREM compositions with multiple modifications are used, then full-length protein expression is restored, but the manufacturing complexity increases
Solution Approach 1:
The TREM composition is divided into distinct functional segments: modified nucleotide regions (2′-O-MOE, pseudouridine, 5,6 dihydrouridine), anticodon domains (ACHD) specific to target PTCs, amino acid acceptance domains (AStD), and structural domains (DH, VL, TH). This segmentation allows modular synthesis and assembly, improving manufacturability while maintaining high expression levels.
Solution Approach 2:
The patent performs preliminary modifications during tRNA synthesis including pre-installation of rare nucleotides and modified bases at specific positions. This preliminary action simplifies downstream processing and ensures proper folding and function, thereby reducing overall manufacturing complexity despite the multiple modifications required for high productivity.
3Reliability
If specific anticodons are designed to pair with PTCs, then read-through efficiency increases, but the design complexity increases
Solution Approach 1:
The anticodon domain (ACHD) of each TREM is locally optimized with specific nucleotide sequences (e.g., UUU for UAG, AAA for UAA, CCU for UGA) that precisely complement target PTCs. This local quality enhancement ensures high read-through efficiency for specific mutations without requiring complex global redesign of the entire tRNA structure, thereby managing design complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The use of TREMs enables the read-through of PTCs, increasing the expression of full-length proteins and maintaining their secondary and tertiary structures, thereby treating diseases associated with truncated proteins.
Implementation Method 1
the TREM, TREM core fragment or TREM fragment has an anticodon that pairs with the codon having the first sequence
Data Source
AI summary
The disclosure relates generally to methods of modulating a production parameter of an RNA corresponding to, or polypeptide encoded by, a nucleic acid sequence comprising an endogenous ORF having a premature termination codon, comprising administering a tRNA-based effector molecule having a non-naturally occurring modification.