Background: 

• TFR1 is a type II transmembrane protein that can bind to transferrin and mediate the uptake of iron ions into cells through endocytosis, thereby regulating intracellular iron homeostasis. Utilizing this endocytosis mechanism, TFR1 can serve as a pathway for mediating the transport of biotherapeutics across the blood-brain barrier. 
• Ids (iduronate-2-sulfatase) is an enzyme that belongs to the family of lysosomal sulfatases. It main functions in glycosaminoglycan metabolism and catabolic pathways. Ids deficiency leads to the accumulation of GAGs in tissues, resulting in a spectrum of metabolic disorders known as mucopolysaccharidosis type II (MPS II or Hunter syndrome). This accumulation can cause various symptoms, such as skeletal abnormalities, cardiovascular issues, and neurocognitive impairments.

Application: 

• The treatment of MPS II targeting Ids faces significant challenges in drug delivery to the brain. To address this challenge, investigating receptor-mediated transport such as through TFR1 can help in facilitating the passage of IDS or its analogs across the blood-brain barrier. So B-hTFR1, Ids KO mice can be used to evaluate the pharmacodynamics and safety of treatments for Ids deficiency related diseases such as MPS II. 

Validation: 

• Human TFR1 protein and RNA were detectable in B-hTFR1 mice. Humanization of TFR1 does not change the overall frequency or distribution of immune cell types in spleen, blood and lymph nodes. Humanization TFR1 does not alter various blood biochemical and hematological parameters.
• Mouse Ids mRNA was only detectable in wild-type C57BL/6 but not in B-hTFR1, Ids KO mice. sGAG was increased in brain and liver of B-hTFR1, Ids KO mice.

Gene targeting strategy for B-hTFR1, Ids KO mice. The exons 4-19 of mouse Tfr1 gene that encode extracellular domain were replaced by human counterparts in B-hTFR1, Ids KO mice. The genomic region of mouse Tfr1 gene that encodes cytoplasmic and transmembrane portion was 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. 

The exons 1-19 of mouse Ids that encode the full length protein were deleted in B-hTFR1, Ids KO mice.

Strain specific analysis of Ids mRNA expression in wild-type C57BL/6 mice and B-hTFR1, Ids KO mice by RT-PCR. Lung and brain RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTFR1, Ids KO mice (H/H, -/-), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Ids primers. Mouse Ids mRNA was only detectable in wild-type C57BL/6 mice but not in B-hTFR1, Ids KO mice. 

Analysis of soluble glycosaminoglycan (sGAG) levels in B-hTFR1, Ids KO mice. Brain (A) and liver (B) tissues were collected from wild-type C57BL/6 mice (+/+) (female, 22-week-old, n=3) and homozygous B-hTFR1, Ids KO mice (H/H, -/-) (female, 22-week-old, n=3) after cardiac perfusion. The tissues were then homogenized and digested, and Blyscan dye was added to form a sulfated glycosaminoglycan-dye complex, which was precipitated, collected, and re-dissolved for ELISA detection. sGAG was increased in brain (A) and liver (B) of B-hTFR1, Ids KO mice, indicting that mouse Ids was knocked out in B-hTFR1, Ids KO mice. Values are expressed as mean ± SEM. 

Select mice aged 3 to 11 weeks, and randomly sample and weigh 5 males and 5 females from each age group. The minimum and maximum weights of the mice in the table are calculated as the average ± SD.