Human Liver Chimeric Animal Model With Por Knockout for Drug Metabolism
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Solution Overview
Problem
Current experimental animal models fail to accurately predict human xenobiotic metabolism, leading to inefficacy or toxicity issues in clinical drug trials, necessitating better preclinical tools.
Innovation Solution
A human liver chimeric non-human animal model is developed by creating a conditional knockout of the NADPH-P450 oxidoreductase (Por) gene in mice, allowing for almost complete replacement with human hepatocytes, using CRISPR/Cas9 and adenoviral gene therapy to inhibit murine cytochrome metabolism.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional animal models are used for drug metabolism studies, then experimental procedures are simple, but prediction accuracy of human drug metabolism is poor
Solution Approach 1:
The patent extracts and removes the murine Por gene through conditional knockout, eliminating the source of murine cytochrome P450 metabolism. This extraction of the problematic genetic element allows human hepatocytes to become the primary metabolic system, thereby improving prediction accuracy while managing model complexity through targeted genetic modification rather than complete species replacement
Solution Approach 2:
The patent applies local quality by creating spatial and functional differentiation within the liver tissue. Human hepatocytes are transplanted to occupy specific liver lobes (preferentially lobes 2 and 3), creating a localized human metabolic system within the mouse organism. This local humanization allows accurate human drug metabolism prediction in specific regions while maintaining the mouse as the overall host system
2Measurement precision
If human hepatocytes are transplanted into immunodeficient mice, then human liver function prediction is improved, but murine cytochrome metabolism interference remains
Solution Approach 1:
The patent applies preliminary anti-action by performing Por gene knockout in the murine liver BEFORE transplanting human hepatocytes. This preemptive elimination of murine cytochrome P450 oxidoreductase prevents murine cytochrome metabolism from occurring in the first place, thereby eliminating interference with human-specific metabolite identification before the human liver system is even established
Solution Approach 2:
The patent performs preliminary action by establishing the Por knockout condition and human hepatocyte transplantation protocol in advance. The mice are genetically prepared with floxed Por alleles and immunodeficient background before human hepatocyte transplantation, ensuring that when human cells are introduced, the murine metabolic interference is already minimized or eliminated
3Object-generated harmful factors
If Por gene is completely knocked out, then murine cytochrome metabolism is eliminated, but embryonic lethality occurs
Solution Approach 1:
The patent segments the Por gene knockout into two distinct phases: (1) Genetic preparation phase where mice are born with floxed Por alleles but maintain normal Por expression and viability, and (2) Induction phase where Cre recombinase is administered to delete Por specifically in adult mice after human hepatocyte transplantation. This segmentation allows the model to be viable during setup while achieving complete Por elimination when needed
Solution Approach 2:
The patent makes the Por gene status dynamic rather than static. Using Cre-loxP system, the Por gene remains intact (floxed) during mouse development and early life, allowing normal development and transplantation procedures. Upon induction with Cre recombinase, the gene is deleted. This dynamic control allows the same animal model to transition from a viable state to a Por-deficient state, combining reliability with complete metabolic elimination
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 model enables accurate prediction of human drug metabolism and toxicity, enhancing the identification of human-specific metabolites and reducing murine interference, thereby improving drug development safety.
Implementation Method 1
The somatic genome engineering comprises Guide RNA (gRNA) and Caspase 9 (Cas9)
Implementation Method 2
The non-human animal can be provided with at least a first dose of a virus that encodes Cre recombinase
Implementation Method 3
The present disclosure solves these needs in the art by providing a human liver chimeric non-human animal model and methods of using the human liver chimeric non-human animal model to predict human specific drug metabolism
Implementation Method 4
providing a non-human animal comprising a deletion of NADPH-P450 oxidoreductase (Por) gene resulting in reduced or absent expression of Por protein
Data Source
Figure 1A
Figure 1B~1C
Figure 1D
AI summary
The present disclosure provides a chimeric non-human animal comprising human hepatocytes, methods for preparing the chimeric non-human animal comprising human hepatocytes and methods of utilizing the chimeric non-human animal comprising human hepatocytes to screening and identifying metabolites for any type of drugs, typically small molecule drugs, which might affect human liver functions and any other bodily function.