Nanobodies as therapies for loss-of-function misfolding diseases: the example of Primary Hyperoxaluria Type 1

Misfolding diseases that result in loss of function represent a considerable burden for both individuals and society. Primary Hyperoxaluria Type 1 (PH1) is a rare genetic disorder caused by the deficit of alanine:glyoxylate aminotransferase (AGT), which leads to excessive endogenous oxalate formation translating into progressive renal damage due to stones formation. The underlying molecular mechanisms causing PH1 are associated with protein misfolding (leading to enhanced degradation, aggregation and/or mitochondrial mistargeting). The main therapeutic approach is a double kidney and liver transplantation, but treatments based on RNA silencing are currently available, although they normalize urinary oxalate excretion only in about 50 % of patients, calling for the need of alternative options. In this work, we developed and characterized a novel biotechnological approach using six single-domain nanobodies (NB-AGT-1 to -6) as potential therapeutics counteracting PH1 misfolding. We show that NB-AGTs are very stable proteins and bind pathogenic and non-pathogenic variants of AGT with high affinities (K_d values from low nM to low pM). Structural studies indicate that NB-AGTs bind to different protein epitopes thus being selective for different variants. Preliminary experiments in a cellular model of PH1 show that internalization of engineered NB-AGT-3 enhances the specific activity of disease-associated variants. Overall, our results provide the basis to consider NBs as a promising approach for PH1 as premise for their application to other loss-of-function misfolding diseases.