BackgroundPatients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD.MethodsSince current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy.ResultsUnder treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells.ConclusionWe conclude that repeated AGAL activity measurements in patients’ white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α‐galactosidase A (AGAL). The formation of neutralising anti‐drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence‐labelled AGAL in combination with ADA‐positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD‐specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre‐incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA‐immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre‐incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient‐derived AGAL‐deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA‐complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.

Migalastat (1-deoxygalactonojirimycin) is approved for the treatment of Fabry disease (FD) in patients with an amenable mutation. Currently, there are at least 367 amenable and 711 non-amenable mutations known, based on an in vitro good laboratory practice (GLP) assay. Recent studies demonstrated that in vitro amenability of mutations did not necessarily correspond to in vivo amenability of migalastat-treated patients. This discrepancy might be due to (methodological) limitations of the current GLP-HEK assay. Currently, there are several published comparable cell-based amenability assays, with partially different outcomes for the same tested mutation, leading to concerns in FD-treating physicians. The aim of this review is to elucidate the idea of amenability assays from their beginning, starting with patient-specific primary cells to high-throughput assays based on overexpression. Consequently, we compare methods of current assays, highlighting their similarities, as well as their pros and cons. Finally, we provide a literature-based list of α-galactosidase A mutations, tested by different assays to provide a comprehensive overview of amenable mutations as a good basis for the decision-making by treating physicians. Since in vitro amenability does not always correspond with in vivo amenability, the treating clinician has the responsibility to monitor clinical and laboratory features to verify clinical response.

The vascular endothelium acts as a selective barrier between the bloodstream and extravascular tissues. Intracellular [Ca 2+ ] i signaling is essential for vasoactive agonist-induced stimulation of endothelial cells (ECs), typically including Ca 2+ release from the endoplasmic reticulum (ER). Although it is known that interactions of Ca 2+ and cAMP as ubiquitous messengers are involved in this process, the individual contribution of cAMP-generating adenylyl cyclases (ACs), including the only soluble AC (sAC; ADCY10), remains less clear. Using life-cell microscopy and plate reader-based [Ca 2+ ] i measurements, we found that human immortalized ECs, primary aortic and cardiac microvascular ECs, and primary vascular smooth muscle cells treated with sAC-specific inhibitor KH7 or anti-sAC-small interfering RNA did not show endogenous or exogenous ATP-induced [Ca 2+ ] i elevation. Of note, a transmembrane AC (tmAC) inhibitor did not prevent ATP-induced [Ca 2+ ] i elevation in ECs. Moreover, Lphenylephrine-dependent constriction of ex vivo mouse aortic ring segments was also reduced by KH7. Analysis of the inositol-1,4,5-trisphosphate (IP 3 ) pathway revealed reduced IP 3 receptor phosphorylation after KH7 application, which also prevented [Ca 2+ ] i elevation induced by IP 3 receptor agonist adenophostin A. Our results suggest that sAC rather than tmAC controls the agonist-induced ER-dependent Ca 2+ response in ECs and may represent a treatment target in arterial hypertension and heart failure.

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