TFR1/APP: The combination of targeted antibody (TFR1) and therapeutic oligonucleotide (APP) 

• Gene Information: The transferrin receptor protein 1 (TFR1) gene encodes human TFR1, a transmembrane glycoprotein. Amyloid precursor protein (APP) gene is located on chromosome 21 with 18 exons and is alternatively spliced into multiple isoforms.
• Protein Expression: TFR1 is highly expressed in the brain endothelial cells of the blood-brain barrier, and can be used as a target for enhancing drug delivery. APP is a cell surface and transmembrane precursor protein that plays a key role in the pathogenesis of Alzheimer's disease. 
• Signaling Pathway: TFR1 primarily mediates the binding and endocytosis of transferrin with its ligand, driving the uptake of iron into cells. Mutations in the APP gene are major contributors to early-onset Alzheimer’s disease and cerebral amyloid angiopathy, and amyloid-beta deposition constitutes one of the hallmark pathological features observed in the brains of patients with Alzheimer’s disease. 
• Therapeutic Inhibition: When conjugated to an oligonucleotide drug targeting human APP, the TFR1-targeting antibody facilitates blood–brain barrier penetration and brain delivery. Reduction of APP protein production is expected to lower the secretion of Aβ peptides that aggregate into extracellular amyloid deposits, as well as diminish APP proteolytic fragments that trigger intraneuronal neurofibrillary tangles and consequently induce neuronal dysfunction characteristic of Alzheimer’s disease.

TFR1 

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 region 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. 

APP 

The partial 5’UTR and CDS that encodes the full-length human APP protein with Swedish (K670N, M671L) and London (V717I) mutation, followed by human 3’UTR-STOP is inserted right after mouse App 5’UTR to replace the exon 1 of mouse App gene. 

The APP protein expression will be driven by endogenous mouse App promoter, while mouse App gene transcription and translation will be disrupted.

Human APP mRNA was exclusively detectable in the cortex and hippocampus of homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice, but not in wild-type mice.

Species-specific analysis of APP gene expression in wild-type C57BL/6J mice and homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice by RT-qPCR. Cortex and hippocampus were collected from wild-type C57BL/6J mice (8-week-old; 3 males and 3 females), homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice (8-week-old, 18-week-old, 3 males and 3 females), then cDNA libraries were synthesized by reverse transcription, followed by PCR with human APP primers. Values are expressed as mean ± SEM.

Mouse App mRNA was exclusively detectable in the cortex and hippocampus of wild-type mice, but not in homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice.

Species-specific analysis of APP gene expression in wild-type C57BL/6J mice and homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice by RT-qPCR. Cortex and hippocampus were collected from wild-type C57BL/6J mice (8-week-old; 3 males and 3 females), homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice (8-week-old, 18-week-old, 3 males and 3 females), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse App primers. Values are expressed as mean ± SEM.

Protein expression analysis of APP and TFR1 in homozygous B-hTFR1/hAPP*K670N*M671L*V717I mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+; 8-week-old), 8-week-old and 18-week-old B-hTFR1/hAPP*K670N*M671L*V717I mice (H/H,H/H), and then analyzed by western blot with anti-APP antibody (Abcam, ab133588) and anti-TFR1 antibody (Abcam, ab214039). 40 μg total proteins were loaded for western blotting analysis. F: female; M: male