Volume 22, Issue 11, Pages 2004-2012 (November 2014) A Chaperone Enhances Blood α-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy  Giancarlo Parenti, Simona Fecarotta, Giancarlo la Marca, Barbara Rossi, Serena Ascione, Maria Alice Donati, Lucia Ovidia Morandi, Sabrina Ravaglia, Anna Pichiecchio, Daniela Ombrone, Michele Sacchini, Maria Barbara Pasanisi, Paola De Filippi, Cesare Danesino, Roberto Della Casa, Alfonso Romano, Carmine Mollica, Margherita Rosa, Teresa Agovino, Edoardo Nusco, Caterina Porto, Generoso Andria  Molecular Therapy  Volume 22, Issue 11, Pages 2004-2012 (November 2014) DOI: 10.1038/mt.2014.138 Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Protocol of ERT and NB-DNJ administration and DBS sampling. (a) The overall duration of the study was 16 months. The patients were subjected to a baseline assessment for two months with rhGAA alone (ERT1), then were treated for 12 months with the combination of rhGAA and NB-DNJ (ERT-CHAP), and subsequently were re-evaluated for two months with rhGAA alone (ERT2). GAA profiling in DBS was performed three times at baseline (ERT1) and three times in the first ERT cycles of ERT+CHAP, and again three times at the end of ERT+CHAP, and three times in ERT2. (b) Patients were treated with rhGAA at standard doses of 20 or 40 mg/kg/infusion (day 0). rhGAA was reconstituted according to the manufacturer's instructions and given intravenously, in 5–7 hours. NB-DNJ was given in four doses of 80 mg/m2 each, one on the evening before ERT (day -1), and three on the day of the infusion (day 0). The sampling for GAA activity in DBS was performed before each ERT infusion (day −1), and on days 1, 3, 5, 7, 9, and 11, (DBS sampling 1) at the beginning of the study (protocol ERT1, months 1–2, and first round of sampling in ERT-CHAP, months 3–4). To better evaluate the GAA profile in the first 72 hours following ERT, at the end of the ERT+CHAP period (months 13–14) and during ERT2 (months 15–16), GAA activities were assayed in DBS every 12 hours for three days (DBS sampling 2). Molecular Therapy 2014 22, 2004-2012DOI: (10.1038/mt.2014.138) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 GAA activity profiles in DBS. (a) The combination of ERT and NB-DNJ (protocol ERT+CHAP) resulted in increases of GAA activity peak areas greater than 1.85-fold, compared to ERT alone in patients 0101, 0103, 0201,0202, 0203, 0204, 0301, 0302, 0303, 0401, 0402). (b) In the whole population, the average GAA activities were significantly increased compared to ERT alone at 12 hours (P = 0.002), 24 hours (P = 0.001), 36 hours (P = 0.003), and remained significantly higher up to 72 hours (inset). The increase in the areas under the curve ranged between 1.90 and 3.42-fold, with an average increase of 6.78, and was also highly statistically significant (P = 0.002). Molecular Therapy 2014 22, 2004-2012DOI: (10.1038/mt.2014.138) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 GAA activities in the Pompe diseasemouse. Pompe disease mice received a single retro-orbital injection of rhGAA with or without an oral administration of NB-DNJ by gavage, and GAA activity was measured in DBS and tissues (liver, quadriceps and gastrocnemius) at 24, 48, and 72 hours. For each time point the average and SD of the activities in three to five animals is shown. The DBS GAA profile in mice showed a maximum peak at 24 hours after the injection and a rapid decrease of activity over the following days (dotted line). A parallel increase in GAA activity was detectable in liver, gastrocnemius and quadriceps, where the GAA activity persisted up to 72 hours (dotted lines). In the animals treated with the combination of ERT and NB-DNJ a significantly higher peak (approximately fivefold compared to ERT alone; P = 0.01 at 24 and 48 hours) was observed in DBS (continuous line). At 48 hours, a statistically significant increase in GAA activity with the combination protocol was observed in liver (P = 0.02), and in quadriceps (P = 0.04). The activities obtained in tissues from the Pompe disease mice treated with ERT alone or with the combination protocol were compared using the Student's t-test. Molecular Therapy 2014 22, 2004-2012DOI: (10.1038/mt.2014.138) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions