Humanized TTR Locus V30M Mutation Animal Model
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Solution Overview
Problem
There is a need for non-human animals that closely mimic human transthyretin (TTR) to enable testing of TTR-targeting reagents and study their efficacy and mode of action, as existing models do not accurately reflect human TTR biology.
Innovation Solution
Creation of non-human animals with a humanized TTR locus comprising a V30M mutation, where regions of the endogenous TTR locus are replaced with corresponding human TTR sequences, allowing for the expression of human TTR protein and facilitating pharmacokinetic and pharmacodynamics studies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing non-human animal models are used, then the models are simpler to create and maintain, but they do not accurately reflect human TTR biology and cannot provide true human target approximation
Solution Approach 1:
The patent applies local quality by replacing only the specific TTR locus region in the non-human animal genome with the human TTR sequence, rather than humanizing the entire genome. This targeted approach maintains the simplicity of the non-human animal model while introducing the specific human TTR biology needed for accurate drug testing. The humanized TTR locus is integrated into the endogenous gene location, ensuring proper expression patterns while preserving the rest of the animal's native genetics.
2Measurement precision
If human TTR sequence is introduced into non-human animals, then accurate human TTR target approximation is achieved, but the creation process becomes more complex requiring precise genomic integration
Solution Approach 1:
The patent employs preliminary action by designing and preparing the human TTR sequence with appropriate flanking regions and regulatory elements before integration. The human TTR cDNA is constructed with 5' and 3' untranslated regions and introns pre-assembled, ensuring proper splicing and expression. This preliminary preparation of the transgene construct facilitates precise integration into the endogenous TTR locus and ensures correct genomic context for accurate pharmacokinetic and pharmacodynamics studies.
3Reliability
If the entire TTR locus is replaced with human sequence, then complete human TTR biology is achieved, but the complexity of genome modification increases significantly
Solution Approach 1:
The patent applies the taking out principle by extracting only the essential human TTR coding sequence and minimal necessary regulatory elements (5' and 3' untranslated regions and introns) required for accurate expression, rather than replacing the entire TTR locus including all regulatory regions. This extracted human TTR sequence is then integrated into the endogenous TTR locus of the non-human animal, achieving complete human TTR biology while simplifying the genome modification process compared to full locus replacement.
Data Source
AI summary
Non-human animal genomes, non-human animal cells, and non-human animals comprising a humanized TTR locus comprising a V30M mutation and methods of making and using such non-human animal genomes, non-human animal cells, and non-human animals are provided. Non-human animal cells or non-human animals comprising a humanized TTR locus express a human TTR protein or a chimeric TTR protein, fragments of which are from human TTR. Methods are provided for using such non-human animals comprising a humanized TTR locus to assess in vivo efficacy of human-TTR-targeting reagents such as nuclease agents designed to target human TTR.