Volume 15, Issue 3, Pages 508-514 (March 2007) Pharmacological Enhancement of Mutated α-Glucosidase Activity in Fibroblasts from Patients with Pompe Disease  Giancarlo Parenti, Alfredo Zuppaldi, M Gabriela Pittis, M Rosaria Tuzzi, Ida Annunziata, Germana Meroni, Caterina Porto, Francesca Donaudy, Barbara Rossi, Massimiliano Rossi, Mirella Filocamo, Alice Donati, Bruno Bembi, Andrea Ballabio, Generoso Andria  Molecular Therapy  Volume 15, Issue 3, Pages 508-514 (March 2007) DOI: 10.1038/sj.mt.6300074 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Effects of imino sugar treatment in Pompe fibroblasts. Treatment with NB-DNJ and DNJ resulted in increased GAA activity in patients 1–4. Mean, SDs, and statistical significance, derived from 4–10 independent experiments for each patient, are indicated. No increase was found in fibroblasts from patients 5–8. NCI, non-classic infantile; J, juvenile; CI, classic infantile. GAA activity in control fibroblasts was 58.5±28.1 nm 4-methylumbelliferone liberated/mg protein/h. Molecular Therapy 2007 15, 508-514DOI: (10.1038/sj.mt.6300074) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Effects of different imino sugar concentrations and time course of GAA enhancement in fibroblasts from patient 1. Enhancement of GAA activity was observed at NB-DNJ concentrations between 10 and 80 μM. The enhancing effect of 20 μM NB-DNJ was already observed after 3 days of incubation and continued up to 15 days. Each point is the mean of two different experiments. Molecular Therapy 2007 15, 508-514DOI: (10.1038/sj.mt.6300074) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Effects of NB-DNJ in HEK293T cells expressing wild-type and mutated GAA. Increase of GAA activity after NB-DNJ treatment was observed in HEK293T cells expressing the mutations L552P and G549R. No increase was observed in cells expressing the mutations A445P and R375L. The specific activities indicated at the top of each bar are the mean of three independent experiments. The activities are expressed as nmoles 4-MU liberated/mg protein/h. Molecular Therapy 2007 15, 508-514DOI: (10.1038/sj.mt.6300074) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Western blot analysis of GAA expression in PD fibroblasts. The figure shows the results obtained in fibroblasts from patients 1, 4 (imino sugar responsive), 7 (non responsive), and from an untreated control cell line. The cells were cultured for 9 days with (+) and without (−) 20 μM NB-DNJ. The arrows indicate the bands corresponding to a precursor peptide of approximately 110 kDa and the mature active molecular forms (76–70 kDa). In all cell lines, an increase of the mature 70–76 kDa GAA molecular forms was observed. The results of Western blot analysis were consistent and reproducible in four independent experiments. Molecular Therapy 2007 15, 508-514DOI: (10.1038/sj.mt.6300074) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Western blot analysis of HEK293T cell extracts expressing single mutations of the GAA gene. After transfection the cells were cultured for 72 h with (+) and without (−) 20 μM NB-DNJ. The arrows indicate the bands corresponding to a precursor peptide of approximately 110 kDa, the intermediate 95 kDa, and the mature active molecular forms (76–70 kDa). In treated cells, an increase of the mature 70–76 kDa GAA molecular forms was observed. Molecular Therapy 2007 15, 508-514DOI: (10.1038/sj.mt.6300074) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Confocal analysis of immunolocalization of GAA in HEK293T expressing L552P-mutated GAA in the absence and presence of NB-DNJ and in cells expressing wild-type GAA. (a) In cells expressing the L552P mutation in the absence of imino sugar treatment, negligible immunofluorescent labeling for GAA was observed. (b) NB-DNJ treatment of the cells resulted in an enhancement of immunofluorescent labeling for GAA with a punctate staining typical of lysosomal localization. (c) This pattern is comparable to that observed in the same cells expressing the wild type enzyme. (d–f) The same cells were stained for an endogenous lysosomal marker (LAMP2). (h, i) In cells overexpressing L552P-mutated GAA in the presence of NB-DNJ, and in cells overexpressing the wild-type enzyme, the fluorescent labeling for GAA partly colocalized, thus confirming an improved delivery of GAA to lysosomes. No colocalization of GAA and LAMP2 signals was observed in untreated HEK293T cells overexpressing 1.522P-mutated GAA (g). Molecular Therapy 2007 15, 508-514DOI: (10.1038/sj.mt.6300074) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions