Targeting Frataxin Dysfunction in Friedreich Ataxia: Nanobody-Based Approaches for Fe-S Cluster Biogenesis Enhancement?

PAVÁN MF; GROSSI, JULIÁN; GENTILI, HERNÁN GUSTAVO; HERMOSO DOMINGUEZ JA; PIGNATARO, MARÍA FLORENCIA; GARAY, ALBA; NOGUERA, MARTÍN EZEQUIEL; RODRIGUEZ N; IBAÑEZ, LORENA ITATI; FERNÁNDEZ, NATALIA BRENDA; MOLINA, RAFAEL; VILA, ANTONELA; ARAN, MARTÍN; SANTOS, JAVIER

Londres

Congreso; International Congress for Ataxia Research (ICAR) 2024; 2024

International Congress for Ataxia Research/Biogen

In Friedreich Ataxia (FA), reduced frataxin (FXN) protein levels lead to cellular dysfunction, primarily by impairingIron-sulfur (Fe-S) cluster production via the ISC machinery. FXN is the kinetic activator of the NFS1-ISCU-ISD11-ACPcomplex, crucial for efficient Fe-S cluster biogenesis. In this work, we propose the quaternary addition of llamananobodies (NB) targeting FXN in order to improve FXN conformational stability and function in cellular models.After llama immunization, we obtained NB libraries, from which anti-FXN NBs were selected by phage display.Interaction was assessed using SEC-FLPC, BLI and NMR. NB candidates were transfected in HEK-293T cells. ISC-NBsinteraction was evaluated by co-immunoprecipitation assays and by measuring the effect over Fe-S related activities.Trojan versions of the NB_4a7 were purified from E. Coli and characterized. We investigated the impact of TrojanNB transduction on mitochondrial metabolism in FRDA fibroblasts through immunofluorescence, mitochondriaisolation and OCR evaluation.NBs showed high levels of expression and mitochondrial localization in HEK-293T and HeLaKyoto cell lines. NBoverexpression in HEK-293T cells did not significantly alter cell viability, Fe-S cluster dependent enzymatic activities(aconitase and Succinate dehydrogenase) nor mitochondria oxygen consumption rates. Also, mature FXN levelswere not significantly altered. We found that NBs interact with FXN and other ISC proteins in a cellular context.TAT_CAMP_4A7 and TAT_COX8_4a7 could be expressed in E.coli. Interestingly, we found that trojan versions of NBsare able to enter FRDA fibroblasts and modulate Fe-S cluster dependent enzymatic activities.Our results suggest that NBs can be expressed and interact with endogenous FXN in a human cell line, withoutaltering Fe-S related activities. Importantly, Trojan NBs can be transduced to FRDA cells, localized in the mitochondria and discretely affect Fe-S related activities. These novel tools may enhance our comprehension of these complex catalytic processes.