The current causal treatment for Fabry disease (FD) is the enzyme replacement therapy (ERT) with recombinant human alpha-galactosidase A (rh-alpha-Gal A), that results in substrate clearance from vascular endothelia and in clinical improvement or stabilization of disease. However, in several patients evidence of disease progression and persistence of Gb3 storage in some cells types, such as podocytes and distal tubular cells has been observed despite enzyme treatment.Werecently demonstrated in fibroblast cell lines of FD patientswith negligible residual enzyme activitya synergistic effect between ERT and the chaperone molecule1-deoxynojirimycin (DGJ).Compared to the effects of rh-alpha-Gal A alone, co-administration of DGJ and rh-alpha-Gal A resulted in better correction (4.8 to 16.9-fold) of intracellular alpha-Gal A activity, increased amounts of the enzyme within the lysosomal compartment, and improved clearance of lyso-Gb3, one of the substrates stored in FD and a potent inhibitor of alpha-Gal A. However, although easy to obta (Fig.1)in and to manipulate in cultures, the fibroblasts are not the preferred site of organ damage and target of therapy in FD.
The aim of our study was to test the combination of the rh-alpha-Gal A with the chaperone molecule DGJ in a cell line of a therapy-target organ of FD, as the cultured renal tubular cells of FD patients with minimal residual enzyme activity.
Renal tubular cells from 3 FD patients were derived from renal biopsies after obtaining the informed consent. All patients presented mutations unresponsive to the chaperone therapy. To study the effect of the small-molecule chaperone DGJ on rh-alpha-Gal A efficacy, the tubular cells were incubated with 5 nmol/l rh-alpha-Gal A for 24 h, in the absence or in the presence of 20 μmol/l DGJ. Untreated cells or cells incubated with DGJ alone were used for comparison. Alpha-Gal A activity was assayed by using the fluorogenic substrate 4-methylumbelliferyl-alpha-D-galactopyranoside (Sigma-Aldrich). Finally, to inhibit the uptake of rh-alpha-Gal A the cells were incubated with the recombinant enzyme in the presence of 5 mmol mannose-6-phosphate (Sigma Aldrich).
None of the cell lines showed significant increases in alpha-Gal A activity when incubated with thechaperonealone, confirming that the GLA gene mutations of these patients were non-responsive to chaperone therapy. The correction of alpha-Gal A activity in the cells incubated with therecombinant enzymealone ranged from 267,41 to 314,37 nmoles 4-methylumbelliferone/mg protein/hour, from 9,78 to 15,02-fold compared to untreated cells. When the cells where co-incubated withDGJ and rh-alpha-Gal Aa highly improved correction of intracellular activity was observed. Increases in alpha-Gal A activity ranged from 38,43 to 59,18-fold, compared to untreated cells (Fig. 2). Compared to the effects of rh-alpha-Gal A alone, co-administration of DGJ and rh-alpha-Gal A resulted in better correction (3,26 to 5,08-fold) ofintracellular alpha-Gal A activity. A representative western blotanalysis of tubular cells from patient 3 (Fig. 3) showed a large increase in alpha-Gal A protein in cells treated with rh-alpha-Gal A and DGJ, compared to the cells treated with rh-alpha-Gal A alone, consistent with the high levels of alpha-Gal A enzyme activity obtained with thecombination therapy.The enhancing effect of DGJ wasnot directed to the endogenous defective alpha-Gal A. First, the cell lines studied were from patients with mutations non-responsive to the chaperone. Second, we observed an increase in the amount of fluorochrome-labelled rh-alpha-Gal A in the presence of DGJ, compared to cells incubated with fluorescent rh-alpha-Gal A alone (Fig. 4): by this approach only the fluorescent exogenous enzyme is detectable and variations in the intensity of fluorescence reflect only the effects on the recombinant enzyme.Finally,the enhancing effect of DGJ on rh-alpha-Gal A was abolished in the presence of mannose-6-phosphate (Fig. 5).
Co-administration of DGJ and rh-alpha-Gal A resulted in all tubular cell lines in a substantial increase in the amount of intracellular enzyme compared with cells treated with rh-alpha-Gal A alone, suggesting improved intracellular stability of the enzyme. In fact, specific interactions between pharmacological chaperones and recombinant enzymes may favor the most stable conformations of the enzymes, protect them in the cells and tissues, and prevent their degradation.
In conclusion, this study provides the first evidence for a synergistic effect between ERT and chaperone therapy on a cell line of a therapy-target organ of FD, and supports the idea that the efficacy of combination protocols may be superior to ERT alone.
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