Humanized RAG/Il2rg Knockout Mice for Granuloma Infection Models
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Solution Overview
Problem
Current genetically modified mice models fail to adequately model certain human pathologies, such as the formation of well-defined granulomas or granulomas containing human immune cells, and do not support the maintenance and propagation of human hematopoietic stem cells, limiting their effectiveness in studying human pathogens like Salmonella typhi and Mycobacterium tuberculosis.
Innovation Solution
Genetically modified mice with RAG and Il2rg gene knockouts and humanization of IL-3, GM-CSF, and optionally TPO genes, engrafted with human hematopoietic cells, capable of forming well-defined granulomas and supporting human immune responses to human pathogens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If genetically modified mice are used to model human immune system, then the ability to study human pathogens is improved, but the accuracy of modeling human pathology (such as granuloma formation) deteriorates
Solution Approach 1:
The patent applies local quality by selectively replacing specific mouse genes (IL-3, GM-CSF, TPO) with their human orthologs in the mouse genome. This creates a localized humanization of the immune system components that are critical for pathogen response, while maintaining the overall mouse physiological background. The targeted gene replacement at specific loci enables accurate modeling of human granuloma formation and immune responses to pathogens like Salmonella typhi and Mycobacterium tuberculosis.
Solution Approach 2:
The patent creates a composite biological system by combining mouse genomic background with human gene insertions. The mouse provides the structural and physiological framework, while human ortholog genes contribute specific immune response capabilities. This composite approach results in a xenogeneic model that exhibits both mouse and human characteristics, enabling accurate modeling of human pathologies while maintaining mouse experimental advantages.
2Reliability
If multiple gene knockouts and humanizations are performed to improve human immune cell engraftment, then the support for human hematopoietic stem cells is improved, but the device complexity increases
Solution Approach 1:
The patent segments the genetic modification process into distinct, manageable components: (1) RAG1 and RAG2 gene knockouts to enable immune cell engraftment, (2) IL-3 gene replacement with human ortholog, (3) GM-CSF gene replacement with human ortholog, and (4) optional TPO gene replacement. Each modification addresses a specific aspect of immune system function or hematopoietic support, allowing systematic optimization without overwhelming complexity.
Solution Approach 2:
The patent employs preliminary action by performing gene knockouts and humanizations during embryonic development or in germline cells, so that the modified mice are born with the desired genetic configuration. This approach establishes the humanized immune system framework before engraftment experiments, eliminating the need for complex post-natal genetic manipulations and reducing overall procedural complexity.
Data Source
AI summary
A mouse with a humanization of the mIL-3 gene and the mGM-CSF gene, a knockout of a mRAG gene, and a knockout of a mIl2rg subunit gene; and optionally a humanization of the TPO gene is described. A RAG/Il2rg KO/hTPO knock-in mouse is described. A mouse engrafted with human hematopoietic stem cells (HSCs) that maintains a human immune cell (HIC) population derived from the HSCs and that is infectable by a human pathogen, e.g., S. typhi or M. tuberculosis is described. A mouse that models a human pathogen infection that is poorly modeled in mice is described, e.g., a mouse that models a human mycobacterial infection, wherein the mouse develops one or more granulomas comprising human immune cells. A mouse that comprises a human hematopoietic malignancy that originates from an early human hematopoietic cells is described, e.g., a myeloid leukemia or a myeloproliferative neoplasia.