Humanized TfR1 and transferrin gene-replacement rats for in vivo evaluation of BBB transport

Abstract

The transferrin receptor 1 (TfR1)-transferrin (TF) axis plays a central role in iron homeostasis and has long been recognized as a promising route for delivering biologics across the blood-brain barrier (BBB). We have developed a class of human-specific anti-TfR1 nanobodies (NewroBus) that exploit this transport pathway. However, the lack of cross-reactivity with rodent TfR1 limits the utility of standard animal models for preclinical testing. To overcome this challenge, we generated knock-in (KI) rats in which the coding sequences of the endogenous Tfrc and Tf genes were replaced with human coding sequences, yielding animals that express human TfR1 and/or human TF under physiological control. Rats homozygous for both humanized alleles were viable and fertile, indicating that the human proteins can functionally replace their rodent equivalents. Nonetheless, these double homozygous rats exhibited erythropoietic abnormalities and tissue-specific alterations in iron distribution--characterized by decreased splenic and increased hepatic iron--suggesting incomplete functional compensation. In contrast, heterozygous rats showed only mild, subclinical hematologic changes (microcytosis and hypochromia). These findings demonstrate that the humanized TfR1-TF axis is compatible with life and iron regulation, albeit with varying degrees of compensation depending on gene dosage. Importantly, these KI rats provide a translationally relevant platform for evaluating pharmacokinetics, CNS penetration, and safety of human-specific BBB-targeting therapeutics, including NewroBus-based biologics and other TfR1-mediated delivery strategies.