A humanized transferrin receptor 1-transferrin model supports functional iron homeostasis and therapeutic delivery across the blood–brain barrier

The transferrin receptor 1 (TfR1)-transferrin (TF) axis is central to iron homeostasis and represents a validated route for delivering biologics across the blood-brain barrier (BBB). We developed human-specific anti-TfR1 nanobodies (NewroBus) that exploit this pathway, but their lack of cross-reactivity with rodent TfR1 limits conventional preclinical testing. To overcome this, we generated knock-in rats in which the coding sequences of the endogenous Tfrc and Tf genes were replaced with their human counterparts, producing animals that express human TfR1 and/or human TF under physiological control. Rats homozygous for both humanized alleles were viable and fertile, indicating functional replacement of their rodent orthologs but exhibited erythropoietic abnormalities and altered iron distribution-reduced splenic and increased hepatic iron-suggesting incomplete compensation. In contrast, heterozygous rats displayed only mild, subclinical microcytosis and hypochromia while maintaining normal BBB integrity and near-physiological iron homeostasis. Using these heterozygous humanized Tfrc rats, we demonstrated that a biologic engineered to engage human TfR1, NewroBus, fused to a therapeutic payload such as TNFα-neutralizing nanobodies, achieved significant BBB penetration and central nervous system exposure. These results validate the translational relevance of this model for studying TfR1-mediated drug delivery. Overall, the humanized TfR1-TF axis is compatible with life and systemic iron regulation, albeit with gene dosage-dependent effects on compensation. These knock-in rats provide a unique, translationally relevant platform for evaluating the pharmacokinetics, central nervous system penetration, efficacy, and safety of human-specific biologics engineered to access the brain via human TfR1-mediated transcytosis.