• TFR1 is a membrane protein essential for iron transport and metabolism, expressed across diverse human tissues. It is markedly upregulated in many cancers, where targeting TFR1 can suppress tumor growth and metastasis. Beyond oncology, TFR1 is also implicated in anemia, neurodegenerative disorders, and other conditions.
• Because TFR1 is highly expressed on brain endothelial cells, it can be leveraged for receptor-mediated transcytosis (RMT), enabling efficient transport of large molecules across the BBB and making it an attractive target for CNS drug delivery.
• Amyloid precursor protein (APP) is a 100-140 kDa transmembrane glycoprotein that plays a key role in the pathogenesis of Alzheimer's disease (AD). In the disease state, β-secretase and γ-secretase aberrantly cleave APP, resulting in the release of amyloid β (Aβ) peptides Aβ40 and Aβ42, which are neurotoxic fragments capable of oligomerizing, aggregating, and subsequently forming plaques.
• Gene editing strategy: B-hTFR1/B-Tg(5XFAD) mice plus  were obtained by mating B-hTFR1 mice (110861) and B-Tg(5XFAD) mice plus (113549).
• mRNA expression analysis: Human APP mRNA was detectable only in B-hTFR1/B-Tg(5XFAD) mice plus but not in wild-type mice.
• Protein expression analysis: Human APP was detected in the cortex and hippocampus from B-hTFR1/B-Tg(5XFAD) mice plus but not in wild-type mice. TFR1 was detected in the heart, liver, spleen, lung, kidney, muscle, cortex, hippocampus, eye and colon from wild-type mice and B-hTFR1/B-Tg(5XFAD) mice plus.
• In vivo efficacy: After administration of oligonucleotide drugs, the human APP mRNA was significantly reduced in the cortex, hippocampus, cerebellum and brain stem, demonstrating that B-hTFR1/B-Tg(5XFAD) mice plus provide a powerful preclinical model for in vivo evaluation of human APP targeted oligonucleotide drugs.
• Application: B-hTFR1/B-Tg(5XFAD) mice plus can be used to assess drug penetration across the blood–brain barrier and screen the oligonucleotide drugs targeting human APP.

Gene targeting strategy for B-hTFR1/B-Tg(5XFAD) mice plus. The exons 4-19 of mouse Tfr1 gene that encode extracellular domain are replaced by human counterparts in B-hTFR1 mice. The genomic region of mouse Tfr1 gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR regions of the mouse gene are also retained. The chimeric TFR1 expression is driven by endogenous mouse Tfr1 promoter, while mouse Tfr1 gene transcription and translation will be disrupted.

B-Tg(5XFAD) mice plus express both mutant human APP with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) familial Alzheimer‘s disease (FAD) mutations and human PSEN1 harboring two FAD mutations, M146L and L286V driven by mouse Thy1 promoter, which is a neural-specific element. And the 5’UTR (except 92 bp was human APP 5’UTR sequence) was the mouse Thy1 counterparts, the 3’UTR regions were replaced with human APP and PSEN1 counterparts.

B-hTFR1/B-Tg(5XFAD) mice plus  were obtained by mating B-hTFR1 mice (110861) and B-Tg(5XFAD) mice plus (113549).

Species specific analysis of mouse App and human APP gene expression in wild-type mice and B-hTFR1/B-Tg(5XFAD) mice plus by RT-qPCR. Cortex and hippocampus RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and B-hTFR1/B-Tg(5XFAD) mice plus, then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with mouse or human APP primers. The expression of mouse App was detected in the cortex and hippocampus of both wild-type mice and B-hTFR1/B-Tg(5XFAD) mice plus, while the expression of human APP was only detected in the cortex and hippocampus of B-hTFR1/B-Tg(5XFAD) mice plus, but not in wild-type mice. The mRNA expression of human APP was significantly higher in the hippocampus compared with the cortex.

Western blot analysis of APP and TFR1 protein expression in B-hTFR1/B-Tg(5XFAD) mice plus. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and B-hTFR1/B-Tg(5XFAD) mice plus (H/H), and then analyzed by western blot with species-specific anti-APP antibody (Abcam, ab133588) and anti-TFR1 antibody (Abcam, Ab214039). 40 μg total protein was loaded for western blotting analysis. Human APP was detected in the cortex and hippocampus from B-hTFR1/B-Tg(5XFAD) mice plus but not in wild-type mice. TFR1 was detected in the heart, liver, spleen, lung, kidney, muscle, cortex, hippocampus, eye and colon from wild-type mice and B-hTFR1/B-Tg(5XFAD) mice plus.

The inhibitory efficiency of the oligonucleotide drugs against human APP in B-hTFR1/B-Tg(5XFAD) mice plus. B-hTFR1/B-Tg(5XFAD) mice plus were randomly divided into four groups (n=3-5/group, 10-week-old, male). The oligonucleotide drugs TA1, TA2, TA3 (provided by client) and vehicle were administered to the mice individually. The mice were sacrificed on day 14, day 35 and day 70, respectively. Then the cortex, hippocampus, cerebellum and brain stem tissue were collected to detect the human APP mRNA by RT-qPCR. (A) The schematic diagram of experimental processing. (B-E) The expression of human APP mRNA in cortex, hippocampus, cerebellum and brain stem. After administration of TA3, the human APP mRNA was significantly reduced compared to the control group (G1), demonstrating that B-hTFR1/B-Tg(5XFAD) mice plus provide a powerful preclinical model for in vivo evaluation of human APP targeted oligonucleotide drugs. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

This experiment was conducted in collaboration with the client using B-hTFR1/B-Tg(5XFAD) mice plus.