• TAU is mainly distributed in the central nervous system, most of it exists in the axons of neurons, and a small amount exists in oligodendrocytes. TAU is involved in neurodegenerative diseases, and most prominently in the pathogenesis of Alzheimer disease (AD).
• TFR1 plays a crucial role in regulating the distribution of iron in the brain, involving multiple biological processes such as cell metabolism, development, myelination, and neurotransmission. Due to the expression of TfR1 in brain endothelial cells, it serves as an ideal target for drug delivery. Various drug delivery strategies targeting TfR1 have been developed to effectively penetrate the blood-brain barrier and promote therapeutic interventions for brain diseases.
• Gene editing strategy: The exons 2~10 of mouse Mapt gene that encode the full-length protein were replaced by human MAPT exons 2~15 in B-hTFR1/hTAU mice. The 3’UTR region of the mouse gene are replaced by human counterparts. The chimeric MAPT expression is driven by endogenous mouse Mapt promoter, while mouse Mapt gene transcription and translation will be disrupted. The exons 4-19 of mouse Tfr1 gene that encode the extracellular region were replaced by human TFR1 exons 4-19 in B-hTFR1/hTAU mice.
• Protein expression analysis: TAU were detected in cortex, hippocampus and cerebellum of both wild-type C57BL/6 mice and homozygous B-hTFR1/hTAU mice. Mouse TFR1 was detectable in wild-type mice. Human TFR1 was exclusively detectable in homozygous hTFR1/hTAU mice but not in wild-type mice.
• In vivo efficacy: Human TAU targeted antibody oligonucleotide conjugates drug was efficacious in B-hTFR1/hTAU mice.
• Application: This product is used for pharmacodynamics and safety evaluation of Alzheimer's disease (AD)

Gene targeting strategy for B-hTFR1/hTAU mice. The exons 2~10 of mouse Mapt gene that encode the full-length protein were replaced by human MAPT exons 2~15 in B-hTFR1/hTAU mice. The 3’UTR region of the mouse gene are replaced by human counterparts. The chimeric MAPT expression is driven by endogenous mouse Mapt promoter, while mouse Mapt gene transcription and translation will be disrupted. The exons 4-19 of mouse Tfr1 gene that encode the extracellular region were replaced by human TFR1 exons 4-19 in B-hTFR1/hTAU mice.

Strain specific analysis of TFR1 and MAPT mRNA expression in wild-type C57BL/6JNifdc and B-hTFR1/hTAU mice by RT-PCR. Cortex RNA was isolated from wild-type C57BL/6JNifdc (+/+) and homozygous B-hTFR1/hTAU mice(H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with TFR1 and MAPT primers. Mouse Tfr1 and Mapt mRNA was exclusively detectable in C57BL/6JNifdc. Human TFR1 and MAPT mRNA was exclusively detectable in homozygous B-hTFR1/hTAU mice.

Species specific analysis of MAPT gene expression in wild-type C57BL/6JNifdc, B-hTAU mice and B-hTFR1/hTAU mice by RT-qPCR. Cortex and hippocampus were collected from wild-type C57BL/6, homozygous B-hTAU mice and homozygous B-hTFR1/hTAU mice (male, 8-week-old, n=3). The mRNA expression of human MAPT in homozygous B-hTAU mice was similar to those in homozygous B-hTFR1/hTAU mice. The mRNA expression of human MAPT was not detectable in wild-type C57BL/6 mice. Values are expressed as mean ± SEM.

Strain-specific TFR1 expression analysis was performed by flow cytometry in homozygous TFR1/TAU humanized mice. Bone marrow and blood cells were collected from wild-type C57BL/6 mice (+/+) and homozygous TFR1/TAU humanized mice (H/H) and analyzed using anti-mouse TFR1 antibody (BioLegend, 113808) and anti-human TFR1 antibody (BioLegend, 334108). Mouse TFR1 was detectable in wild-type mice, whereas human TFR1 was exclusively detectable in homozygous TFR1/TAU humanized mice and not in wild-type mice.

Western blot analysis of TFR1 and TAU protein expression in homozygous B-hTFR1/hTAU mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice, B-hTAU mice, B-hTFR1 mice and homozygous B-hTFR1/hTAU mice (male, 8-week-old), and then analyzed by western blot with anti-TFR1 antibody (Abcam, ab214039) and anti-TAU antibody (CST, 46687). 40 μg total protein was loaded for western blotting analysis. TFR1 was detected in cortex, hippocampus, cerebellum and heart of wild-type C57BL/6JNifdc mice, B-hTAU mice, B-hTFR1 mice and homozygous B-hTFR1/hTAU mice. TAU was detected in cortex, hippocampus and cerebellum of wild-type C57BL/6JNifdc mice, B-hTAU mice, B-hTFR1 mice and homozygous B-hTFR1/hTAU mice.

The inhibitory efficiency of the antibody oligonucleotide conjugate (AOC) drug against human MAPT in B-hTFR1/hTAU mice. B-hTFR1/hTAU mice were randomly divided into three groups (n=3/group, 7-week-old, female). The vehicle, antibody oligonucleotide conjugates drug and oligonucleotide drug were administered to B-hTFR1/hTAU mice individually on day 0 and day 7. The mice were sacrificed on day 21, and the hippocampus, cortex and spinal cord were collected to detect the expression level of human MAPT mRNA by qPCR. The human MAPT mRNA in the treatment group (antibody oligonucleotide conjugates drug) were significantly reduced compared to the control groups (oligonucleotide drug and vehicle) in the hippocampus, cortex and spinal cord, demonstrating that B-hTFR1/hTAU mice provide a powerful preclinical model for in vivo evaluation of human MAPT targeted antibody oligonucleotide conjugates drug. (A) The schematic diagram of experimental processing. (B) The expression of human MAPT mRNA in hippocampus, cortex and spinal cord. Values are expressed as mean ± SEM. Significance was determined by unpaired t test. *P < 0.05.

This experiment was conducted in collaboration with the client using B-hTFR1/hTAU mice.