Humanized Coagulation Factor XII Locus in Non-Human Animals
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Solution Overview
Problem
There is a need for non-human animals that closely mimic human coagulation factor XII (F12) to enable effective testing of F12-targeting reagents, including assessing their efficacy and mode of action, as well as conducting pharmacokinetic and pharmacodynamics studies.
Innovation Solution
The development of non-human animals with a humanized coagulation factor XII locus, where a segment of the endogenous F12 locus is replaced with a corresponding human F12 sequence, allowing for the expression of human coagulation factor XII proteins or chimeric proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a non-human animal model is used to test human F12-targeting reagents, then testing can be performed in live animals, but the model does not accurately reflect human F12 biology
Solution Approach 1:
The patent applies local quality by replacing only the F12 gene locus in the non-human animal genome with the human F12 sequence, while keeping the rest of the genome as the original species. This creates a humanized F12 model that accurately reflects human F12 biology at the specific locus of interest while maintaining the physiological context of the host species.
Solution Approach 2:
The patent uses a non-human animal as an intermediary system that bridges the gap between in vitro human cell studies and direct human clinical trials. The humanized F12 locus in the non-human animal serves as a mediator that allows testing of human F12-targeting reagents in a live whole-organism system while still being genetically compatible with human F12 biology.
2Manufacturing precision
If the entire F12 locus is replaced with human sequence, then human F12 expression is achieved, but genomic integration and regulation may be disrupted
Solution Approach 1:
The patent uses preliminary action by employing CRISPR/Cas9 genome editing technology to create a targeted double-strand break at the endogenous F12 locus before introducing the human F12 sequence. This pre-prepared genomic site ensures accurate integration of the human sequence at the correct location, maintaining genomic stability while achieving human F12 expression.
Solution Approach 2:
The patent copies the human F12 locus sequence and integrates it into the endogenous F12 location in the non-human animal genome. This copying approach ensures that the human F12 sequence is precisely replicated and inserted at the appropriate genomic position, maintaining the structural and functional integrity of the genome while achieving human-specific F12 expression.
Data Source
AI summary
Non-human animal genomes, non-human animal cells, and non-human animals comprising a humanized coagulation factor XII (F12) locus 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 F12 locus express a human coagulation factor XII protein or a chimeric coagulation factor XII protein, fragments of which are from human coagulation factor XII. Methods are provided for using such non-human animals comprising a humanized F12 locus to assess in vivo efficacy of human-coagulation-factor-XII-targeting reagents such as nuclease agents designed to target human F12. A short isoform of F12 that is produced locally in the brain, and methods of using the short isoform, are also provide.