Multiplex Humanization: Models like B6-hCD47/hSIRPα demonstrate 4.7-fold increased tumor clearance with dual blockade.
Conditional Systems: Tissue-specific co-expression (e.g., Tet-On) ensures <2% leaky expression.

High-Fidelity Editing: eSpCas9(1.1) reduces off-target effects by >95%.
ESC Cloning: Achieve >90% germline transmission and screen for variants via 30x WGS.

Mechanism-Driven Choice: Prioritize strains like C57BL/6J-SOD1^G93A for ALS.
Genetic Purity: Backcross humanized alleles into >98% pure backgrounds.
Phenotypic Screening: Pre-screen strains to identify clinically relevant modifiers.

Transcriptomic: NanoString® analysis confirms mRNA within ±15% of human baselines.
Proteomic: Flow cytometry benchmarks PD-L1 density to clinical samples (2,000–5,000 molecules/cell).
Functional: Anti-PD-1 response correlation of R²=0.78 with clinical data.

Off-Target Effects: B6-hCD47 models showed <5% off-target lysis of CD47+ stromal cells (7-AAD/Annexin V assays).
CRS Correlation: Humanized IL6/IL6R models achieved 89% alignment between murine cytokine spikes and clinical CRS grading.

Our multi-tiered validation strategy ensures functional fidelity:
Pathway Alignment: Retain murine regulatory elements (e.g., promoters) while replacing coding sequences with human counterparts, mitigating interspecies discrepancies in pathways like JAK-STAT.
Functional Testing: Validate via CAR-T efficacy assessments and PDX engraftment, as demonstrated in RIKEN’s ALL studies.
Receptor-Ligand Compatibility: Co-humanize interacting pairs (e.g., PD-1/PD-L1) to preserve binding affinity.