An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model

Abstract

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.

A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study

“…For that reason, we tested whether a different set of gelsolin nanobodies could be applied as a chaperone for gelsolin to divert furin activity in the same manner as we had implemented the FAF Nb1-3 towards C68 [9].…”

“…It is therefore important, during therapy development, to test promising compounds over a long period of time and assess the effects at different time points during the course of the trial. During the Nb11 experiments, this had to be done by using different groups for every time point since the current methods of AGel analysis are end-stage [8,9]. Not only does this greatly augment the amount of mice needed, it also makes it impossible to evaluate the drug effect and amyloid build-up in a continuous manner.…”

“…In a first approach, nanobodies, which partly protect C68 against MT1-MMP, were intraperitoneally injected in AGel mice [8]. In a second approach, a mouse model expressing Nb11 was developed [9]. Nb11 binds to mutant plasma gelsolin (PG*) and shields it from furin degradation.…”

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

“…Currently, there are no nanobody-based products approved as therapeutic agent, but several products are in the pipeline or in advanced clinical trials. Their value in treating envenoming [21], infections [22], amyloidosis [23,24], cancer, and other pathologies [25,26] has already been proven. The research concerning the use of nanobodies as a therapeutic agent is mainly performed on extracellular targets.…”

“…A pathological proteolytic cascade involving furin and MT1-MMP like proteases leads to the secretion of amyloidogenic 8 and 5 kDa peptides in the extracellular matrix and thus to the formation of extracellular deposits [63]. Van Overbeke et al used gelsolin nanobodies to shield mutant plasma gelsolin (PG) from aberrant furin cleavage [24]. Furin is a membrane-associated pro-protein convertase that is ubiquitously expressed.…”