pH-dependent anti-TfR1 NANOBODY(R) molecules deliver efficacious oligonucleotide payloads to muscle and CNS tissues

Hoyt, E. A.; Moonens, K.; Rapisarda, C.; Salvador, A. M.; Hammond, T. R.; Kharade, S.; Mondragon Gonzalez, R.; Moran, F.; Ramkumar, S.; Thummapudi, J.; Zhou, S.; Haussy, G.; Capdevila, C.; Maillard, F.; Ismail, A.; Avery, L.; Sardi, P.; Nonne, C.; Cornelis, S.; Leksa, N.

doi:10.64898/2026.07.29.741483

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bioRxiv · 10.64898/2026.07.29.741483

pH-dependent anti-TfR1 NANOBODY(R) molecules deliver efficacious oligonucleotide payloads to muscle and CNS tissues

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Abstract

The blood-brain barrier (BBB) is a highly selective, semi-permeable border of endothelial cells that prevents solutes and therapeutic agents in systemic circulation from passively crossing into the central nervous system (CNS) parenchyma. The Transferrin receptor 1 (TfR1) endocytosis pathway for iron homeostasis is one of the most well-characterized strategies for therapeutic delivery across the BBB. The work presented here showcases the discovery of novel anti-TfR1 NANOBODY(R) shuttles. The identified anti-TfR1 NANOBODY(R) molecules display cross-reactivity and pH-dependent binding to human, cynomolgus (cyno), and mouse TfR1. Structural data further explain and support the underlying mechanism of this pH-dependent binding. These anti-TfR1 NANOBODY(R) molecules were successfully conjugated to both short-interfering RNA (siRNA) and antisense oligonucleotide (ASO) tool payloads. anti-TfR1 NANOBODY(R)-siRNA conjugates can induce up to 60% knockdown of the target mRNA transcript in skeletal muscle up to two weeks post a single IV dose in mice and up to 35-40% at four weeks post dose. Furthermore, extending the half-life of the anti-TfR1 NANOBODY(R)-ASO shuttles enhances heart, sciatic nerve, and brain exposure and enables up to 30-60% target knockdown in different CNS cell types. Altogether, these results highlight important features for the development of anti-TfR1 shuttles for the purpose of downregulating target mRNA transcripts in muscle and CNS for a variety of neurologic and neuromuscular indications.

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Hoyt, E. A., Moonens, K., Rapisarda, C., Salvador, A. M., Hammond, T. R., Kharade, S., Mondragon Gonzalez, R., Moran, F., Ramkumar, S., Thummapudi, J., Zhou, S., Haussy, G., Capdevila, C., Maillard, F., Ismail, A., Avery, L., Sardi, P., Nonne, C., Cornelis, S., Leksa, N.. 2026-08-03. pH-dependent anti-TfR1 NANOBODY(R) molecules deliver efficacious oligonucleotide payloads to muscle and CNS tissues. https://doi.org/10.64898/2026.07.29.741483

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