The Pharmacological Chaperone AT2220 Increases Recombinant Human Acid α-Glucosidase Uptake and Glycogen Reduction in a Mouse Model of Pompe Disease

Abstract

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.

Full text

The Pharmacological Chaperone AT2220 Increases
Recombinant Human Acid a-Glucosidase Uptake and
Glycogen Reduction in a Mouse Model of Pompe Disease
Richie Khanna*, John J. Flanagan, Jessie Feng, Rebecca Soska, Michelle Frascella, Lee J. Pellegrino,
Yi Lun, Darlene Guillen, David J. Lockhart, Kenneth J. Valenzano
Amicus Therapeutics Inc, Cranbury, New Jersey, United States of America
Abstract
Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid a-glucosidase
(GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles.
Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of
Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical
manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some
key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-
administration of the orally-available, small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochlo-
ride, duvoglustat hydrochloride) may improve the pharmacological properties of rhGAA via binding and stabilization.
AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37uC in both buffer and
blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life
of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in
significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles,
compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of
AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-
administered AT2220 on rhGAA in Pompe patients.
Citation: Khanna R, Flanagan JJ, Feng J, Soska R, Frascella M, et al. (2012) The Pharmacological Chaperone AT2220 Increases Recombinant Human Acid a-
Glucosidase Uptake and Glycogen Reduction in a Mouse Model of Pompe Disease. PLoS ONE 7(7): e40776. doi:10.1371/journal.pone.0040776
Editor: David R. Borchelt, University of Florida, United States of America
Received March 30, 2012; Accepted June 13, 2012; Published July 18, 2012
Copyright: ß2012 Khanna et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: No external funding was used for this study.
Competing Interests: All authors are full-time employees and shareholders of Amicus Therapeutics. The authors have read and adhere to all PLoS ONE policies
on sharing the data and materials in this study.
* E-mail: rkhanna@amicusrx.com
Introduction
Pompe disease (OMIM 23200), also referred to as glycogen
storage disease type II or acid maltase deficiency, is a lysosomal
storage disease (LSD) caused by mutations in the gene (GAA)
that encodes the lysosomal hydrolase acid a-glucosidase (GAA)
[1–2]. Deficiency of GAA activity results in progressive
accumulation and deposition of glycogen in the lysosomes of
heart, skeletal muscles, and other tissues. The disease encom-
passes a broad spectrum of phenotypes that range from the
severe infantile-onset form to more slowly progressing late-onset
forms [2–5]. Early or infantile-onset Pompe disease occurs
before 12 months of age, has a rapid rate of progression, and is
typically characterized by severe muscle weakness, frequent
respiratory infections, hepatomegaly, massive cardiomegaly,
cardiomyopathy, and cardiorespiratory failure that usually
results in death between 1 and 2 years of age [1,3]. Late-
onset forms of the disease occur between childhood and
adulthood, have a slower rate of progression, and are typically
characterized by musculoskeletal and pulmonary involvement
that lead to progressive weakness and respiratory insufficiency
[1,3–5].
Enzyme replacement therapy (ERT) is currently the primary
treatment for Pompe disease [6]. ERT is based on the intravenous
administration of recombinant human GAA (rhGAA), of which
MyozymeHand LumizymeH(alglusidase alfa; Genzyme Corpora-
tion, Cambridge, MA) are the only two approved products. Infantile
Pompe patients treated with ERT show improvements on cardiac
hypertrophy and motor skills, with a substantial increase in life-span
[5,7–9]. Late-onset patients have shown mild improvements in
motor and respiratory functions following therapy with ERT,
though clinical efficacy in these patients still requires long-term
evaluation [10–11]. Despite the clinical benefits of ERT, a number
of reports suggest that correction of the skeletal muscle phenotype is
particularly challenging, and that not all patients respond equally
well to treatment [2,7,12–13]. These limitations are at least partially
due to insufficient targeting/uptake into disease-relevant tissues, as
well as poor tolerability due to severe ERT-mediated anaphylactic
and immunologic reactions [5,11,14–18].
Small molecule pharmacological chaperones (PCs) have been
proposed as a potential therapy for Pompe disease [19–22]. The
iminosugar 1-deoxynojirimycin hydrochloride (AT2220, duvoglu-
stat hydrochloride) acts as a PC for GAA, selectively and reversibly
binding and stabilizing the endogenous enzyme, facilitating proper
PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40776

Figure 1. AT2220 increases the physical stability of rhGAA in vitro. (A) Time course of rhGAA denaturation in neutral and acidic buffer at
37uC in the absence and presence of 50 mM AT2220. Denaturation was monitored by changes in the fluorescence of SYPRO Orange as a function of
time. (B) Time course of rhGAA inactivation (i.e. loss of activity) in neutral and acidic buffer at 37uC in the absence and presence of 50 mM AT2220. (C)
Time course of rhGAA inactivation (i.e. loss of activity) in human whole blood at 37uC in the absence and presence of 50 mM AT2220. In both (B) and
(C), GAA enzyme activity was determined at the indicated time points using the fluorogenic substrate 4-MUG. To obtain relative enzyme activity
levels, measurements at the various time points were compared to the activity at the zero time point.
doi:10.1371/journal.pone.0040776.g001
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40776

protein folding and trafficking to lysosomes [21]. AT2220 has been
shown to increase endogenous levels of many different mutant forms
of GAA [21–22]. PCs have also been identified that selectively bind,
stabilize, and increase the levels of the mutated enzymes that are
associated with several other LSDs, including Gaucher, Tay-Sachs,
Sandhoff, GM1-gangliosidosis, and Fabry disease [23].
Recently, the ability of PCs to improve the physical stability,
and to increase cellular and tissue uptake, has been demonstrated
for several exogenous recombinant enzymes that are used to treat
LSDs. Specifically, the PCs AT1001 (deoxygalactonojirimycin),
isofagomine, and N-butyl-deoxynojirimycin were shown to
increase the in vitro cellular uptake and/or in vivo tissue uptake of
the recombinant enzymes used to treat Fabry [24–26], Gaucher
[27], and Pompe [24] diseases, respectively. Furthermore, AT1001
co-administration with recombinant human a-galactosidase A
leads to greater substrate reduction in cells and tissues of Fabry
mice compared to administration of enzyme alone [25–26].
Here, we demonstrate that AT2220 stabilizes rhGAA in vitro,
minimizing protein denaturation and inactivation at neutral pH
and physiological temperature. Studies in rats and GAA knock-out
mice indicate that oral pre-administration of AT2220 increases the
circulating half-life of rhGAA, and leads to significant increases in
rhGAA activity in disease-relevant tissues. Most importantly, we
show that AT2220-mediated increases in rhGAA tissue levels
translate to greater glycogen reduction compared to administra-
tion of rhGAA alone, thus indicating a ‘‘boost’’ in the net
lysosomal activity from the exogenous recombinant enzyme.
Taken together, these data indicate that AT2220 can increase
the stability and improve the pharmacokinetic properties of
rhGAA, thereby leading to increased tissue enzyme activity and
greater substrate reduction. As such, a Phase 2 clinical study has
been initiated to investigate the effects of co-administered AT2220
on rhGAA in Pompe patients.
Materials and Methods
Materials
All cell culture reagents were purchased from Invitrogen
(Carlsbad, CA), except for characterized fetal bovine serum
(FBS), which was purchased from HyClone (Waltham, MA).
AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydro-
chloride) was synthesized by WuXi PharmaTech (Shanghai,
China). Recombinant human acid a-glucosidse (rhGAA; algluco-
sidase alfa; MyozymeH) was purchased from Genzyme Corp.
(Cambridge, MA). The rabbit anti-human GAA polyclonal
antibody, FL059, was a kind gift of Dr. Barry Byrne (University
of Florida, Gainesville). Horseradish peroxidase-conjugated goat
anti-rabbit IgG secondary antibody was purchased from Thermo-
Pierce (Jackson Immunosearch Labs, West Grove, PA). GAA
knock-out (KO) mice were kindly provided by Dr. Barry Byrne.
Wild-type C57BL/6 mice and Sprague-Dawley rats (carotid artery
cannulated) were purchased from Taconic Farms (Germantown,
NY). Animal husbandry and all experiments were conducted
under Institutional Animal Care and Use Committee approved
protocols. All other reagents were purchased from Sigma Aldrich
(St. Louis, MO) unless noted otherwise.
Thermal Stability
The stability of rhGAA was assessed using a modified fluorescence
thermostability assay [28] on a Realplex Mastercycler qRT-PCR
system (Eppendorf, Hamburg, Germany) in either neutral pH buffer
(25 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) or
acidic pH buffer (25 mM sodium acetate, 150 mM sodium chloride,
pH 5.2). For Fig. 1A, rhGAA (2.5 mg) was combined with SYPRO
Orange and 50 mM AT2220 in a final reaction volume of 25 mL. For
time-dependent denaturation assay, reactions were incubated at
37uC for up to 24 hours, with SYPRO Orange fluorescence intensity
monitored at the indicated time points.
Enzyme Inactivation in Buffer or Whole Blood
For time-dependent loss of activity assays shown in Figs. 1B
and 1C, rhGAA (500 nM) was incubated with or without 50 mM
AT2220 for 15 minutes on ice in various pH buffers (neutral or
acidic) or whole blood (Lampire; Pipersville, PA). Reactions were
then transferred to 37uC, with aliquots removed at the indicated
time points and measured for GAA activity. For GAA activity
measurements, samples were diluted 100-fold in 50 mM potassi-
um acetate (pH 4.0) prior to a further 10-fold dilution into a 96-
well plate containing reaction buffer (50 mM potassium acetate,
3.3 mM 4-methylumbelliferryl-a-D-glucopyranoside (4-MUG),
pH 4.0). Activity assays were performed for one hour at 37uC
and stopped by the addition of an equal volume of 0.5 M sodium
carbonate (pH 10.5). Liberated 4-MU was measured on a Victor
3
plate reader (Perkin Elmer, Waltham, MA) at 355 nm excitation
and 460 nm emission. Normalized fluorescence data were plotted
as a function of time.
AT2220/rhGAA Co-administration Studies in Rats
The rat co-administration studies were designed to investigate
the effects of 3 and 30 mg/kg AT2220 on the circulating half-life
of 10 mg/kg rhGAA when administered as a bolus tail vein
injection or as an intravenous infusion. This dose of rhGAA
provided significant activity above the endogenous GAA levels in
normal rat plasma, and minimized the quantity of rhGAA
required to complete these studies. Furthermore, 3 and 30 mg/
kg AT2220 yield plasma exposures in rats that are comparable to
those seen in humans following oral administration of 50 and
600 mg AT2220, respectively [29]. For IV bolus study (Fig. 2A),
eight-week old male Sprague-Dawley rats were administered
either vehicle (water) or AT2220 (3 or 30 mg/kg) via oral gavage.
Thirty minutes later, vehicle (saline) or rhGAA (10 mg/kg) was
administered via bolus tail vein injection. Whole blood was
collected into lithium heparin tubes from the carotid artery
cannula at the indicated time points. Plasma was collected by
centrifuging blood at 2700 g for 10 minutes at 4uC, and was used
for measurement of GAA activity as described below. For the
infusion study (Fig. 2B), the same procedure was followed, except
that rhGAA was administered as an intravenous infusion over 60
minutes at a rate of 5 mL/kg/hour. Comparative descriptions of
the various doses, routes, and regimens for rhGAA and AT2220
administration are presented in Table 1.
AT2220/rhGAA Co-administration Studies in GAA KO
Mice
Studies in GAA KO mice were designed to investigate the effects
of a higher, clinically-relevant dose of rhGAA (20 mg/kg), as the
focus was primarily efficacy (measured by rhGAA tissue uptake
and glycogen reduction). The doses of AT2220 were limited to 10
and 30 mg/kg, which yield plasma exposures in mice that are
comparable to those seen in humans following oral administration
of 200 and 600 mg AT2220, respectively [29]. Our unpublished
data suggest that even larger doses of AT2220 result in high and
sustained muscle concentrations that have the potential for long-
term inhibition of rhGAA; hence, doses above 30 mg/kg were not
investigated [29]. In the studies shown in Figs. 3, 4, 5, and 6, 12-
week old male GAA KO mice were administered vehicle (water) or
AT2220 (10 and/or 30 mg/kg) via oral gavage once every other
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40776

Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40776

week for 8 weeks. Thirty minutes after AT2220, rhGAA (20 mg/
kg) was administered via bolus tail vein injection. Diphenhydra-
mine (DPH; 10 mg/kg) was administered intraperitoneally 10
minutes before the 3
rd
and 4
th
rhGAA injections to minimize
hypersensitivity reactions to rhGAA. Tissues were collected 7 or
21 days following the last rhGAA administration. In all studies,
heart, diaphragm, tongue, skin (shaved and removed from the
lower ventral side of the neck), hindlimbs (for isolation of
quadriceps and gastrocnemius), and forelimbs (for isolation of
triceps) were quickly removed, rinsed in cold phosphate-buffered
saline, blotted dry, and stored on dry ice. Samples of heart and
quadriceps were stored in fixatives as described below for
immunohistochemical analysis. Comparative descriptions of the
various doses, routes, and regimens for rhGAA and AT2220
administration are presented in Table 1.
Measurement of Plasma GAA Levels
For plasma GAA activity measurements, samples were diluted
400-fold with Lysis Buffer (1% Triton X-100, 150 mM NaCl,
25 mM Bis-Tris, pH 6.5) prior to assay. Plasma (20 mL) was added
to 50 mL Assay Buffer (3 mM 4-MUG in 50 mM potassium
acetate, pH 4.0) and incubated for 1 hour at 37uC. Reactions were
stopped by addition of 70 mL 0.4 M glycine, pH 10.8. Fluores-
cence at 460 nm was read on a Victor
3
plate reader after
excitation at 355 nm. Raw fluorescence counts were background
subtracted (defined by Assay Buffer only). A 4-MU standard curve
ranging from 7 nM to 15 mM was run each day for conversion of
fluorescence counts to absolute GAA activity, expressed as
nanomoles of released 4-MU per milliliter of plasma per hour
(nmol/mL/hr). For Western blotting, diluted plasma (,10 mg
total protein) was subjected to SDS-PAGE on 12% polyacrylamide
gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes
(Bio-Rad), and immunoblotted with the rabbit anti-human GAA
primary antibody FL059 (1:1000 dilution). Protein bands were
detected using peroxidase-conjugated goat anti-rabbit secondary
antibody in combination with enhanced chemiluminescence
(Pierce, Rockford, IL). Blots were scanned on an Image Station
4000R (Kodak, Rochester, NY).
Measurement of Tissue GAA Levels
Tissue lysates were prepared by homogenization of ,50 mg
tissue for 3 to 5 seconds on ice with a micro-homogenizer (Pro
Scientific, Thorofare, NJ) in 200 mL Lysis Buffer. Lysates (20 mL)
Figure 2. AT2220 increases the circulating half-life of rhGAA in rats. (A) Eight-week old male Sprague Dawley rats were administered vehicle
(water) or AT2220 (3 or 30 mg/kg) via oral gavage. Thirty minutes later, vehicle (saline) or rhGAA (10 mg/kg) was administered via bolus tail vein
injection. Blood was collected at the indicated time points, and GAA activity (upper panel) and protein levels (lower panel) were measured in plasma
as described in ‘Materials and Methods’. (B) Eight-week old male Sprague Dawley rats were administered vehicle (water) or AT2220 (3 or 30 mg/kg)
via oral gavage. Thirty minutes later, vehicle (saline) or rhGAA (10 mg/kg) was administered via 60-minute intravenous infusion. Blood was collected
at the indicated time points, and GAA activity (upper panel) and protein levels (lower panel) were measured in plasma. PS: post-start infusion; PE:
post-end infusion. In both (A) and (B), each time point represents the mean6SEM of the activity measured from 3 rats; each lane on the Western blot
contains plasma from a single rat, and is representative of two rats in each group.
doi:10.1371/journal.pone.0040776.g002
Table 1. Description of the different doses, routes, and regimens utilized for rhGAA and AT2220 administration.
Figure Species Age (weeks) rhGAA (mg/kg)
AT2220
(mg/kg)
Route of Administration/
Timing Number of Administrations
2A SD rats 8 10 3 and 30 AT2220 PO 30 minutes prior to
rhGAA IV bolus
Single
2B SD rats 8 10 3 and 30 AT2220 PO 30 minutes prior to
rhGAA IV infusion
Single
3, 4, and 6 GAA KO mice 12 20 30 AT2220 PO 30 minutes prior to
rhGAA IV bolus
Four bi-weekly
5GAA KO mice 12 20 10 and 30 AT2220 PO 30 minutes prior to
rhGAA IV bolus
Four bi-weekly
SD, Sprague Dawley.
doi:10.1371/journal.pone.0040776.t001
Figure 3. AT2220 increases the circulating levels of rhGAA in
GAA KO mice. Twelve-week old male GAA KO mice were administered
vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other
week for 8 weeks. Thirty minutes after each AT2220 oral administration,
vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail
vein injection. Blood was collected after the last (i.e., 4
th
) rhGAA
administration and, GAA activity (upper panel) and protein levels (lower
panel) were measured in plasma as described in ‘Materials and
Methods’. Each bar represents the mean6SEM of the GAA activity
measured from 5 mice per group. Statistically significant increases were
seen in plasma GAA activity compared to baseline (*p,0.05, t-test) and
compared to rhGAA administration alone (#p,0.05, t-test). Each lane
on the Western blot contains plasma from a single mouse, and is
representative of two mice in each group.
doi:10.1371/journal.pone.0040776.g003
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40776

were added to 50 mL Assay Buffer as described above. A Micro
BCA Protein Assay (Pierce) was used to determine total protein
concentration in tissue lysates according to the manufacturer’s
instructions. A 4-MU standard curve was run each day, and GAA
activity was measured as described above and is expressed as
nanomoles of released 4-MU per milligram of total protein per
hour (nmol/mg protein/hr).
Measurement of Tissue Glycogen Levels
Tissue glycogen levels were measured as described previously
[30] with slight modifications. Briefly, tissue lysates from GAA
KO and wild-type mice were prepared by homogenization of
,50 mg tissue for 3 to 5 seconds on ice with a micro-
homogenizer in 200 mL Lysis Buffer. Lysates were heat
denatured at 99uC for 10 minutes and centrifuged for 10
minutes at 4uC. Supernatants from GAA KO and wild-type mice
were diluted 1:10 and 1:2, respectively, with Lysis Buffer. Diluted
lysates (40 mL) were incubated in duplicate with or without
10 mL of 800 U/mL amyloglucosidase for 1 hour at 50uC. The
reactions were stopped by heat inactivation at 99uC for 10
minutes, followed by cooling at 4uC for 1 hour. Finally, 200 mL
Glucose Assay Reagent was added and absorbance at 340 nm
was measured on a Spectramax M2e (Molecular Devices;
Sunnyvale, CA). A glycogen standard curve ranging from 5 to
400 mg/mL was run each day for conversion of absorbance data
to absolute glycogen levels. Protein levels were measured in
lysates (before denaturing) using the Micro BCA Protein Assay
Kit according to the manufacturer’s instructions. Data were
Figure 4. Co-administration of AT2220 promotes greater tissue uptake of rhGAA in GAA KO mice. Twelve-week old male GAA KO mice
were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral
administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 7 days after the last (i.e.,4
th
)
rhGAA administration and tissue GAA activity was measured as described in ‘Materials and Methods’. Each bar represents the mean6SEM of the GAA
activity measured from 5 mice per group. Statistically significant increases were seen in GAA activity compared to baseline (*p,0.05, t-test) and
compared to rhGAA administration alone (#p,0.05, t-test). For comparison, GAA levels in wild-type C57BL/6 mice were 1562, 1660.6, 2163, 1862,
1162, and 2563 nmol/mg protein/hr in heart, diaphragm, gastrocnemius, quadriceps, triceps, and tongue, respectively (mean6SEM of 7 mice).
doi:10.1371/journal.pone.0040776.g004
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40776

expressed as micrograms of glycogen cleaved/milligram of
protein (mg/mg protein).
Histochemical Detection of Glycogen
Glycogen levels in heart and quadriceps were determined
histochemically using two different types of tissue processing and
PAS staining methods. For heart, tissues were fixed, processed,
and embedded in Epon-Araldite according to the protocol
described previously [31]. Staining was performed using a Periodic
Acid-Schiff (PAS) Kit (Sigma) according to the manufacturer’s
instructions. Briefly, 1 mm sections were cut and mounted on
slides. Sections were hydrated in deionized water for 5 minutes
before immersion in 1% periodic acid for 5 minutes. After a brief
rinse in deionized water, slides were left in Schiff’s reagent for 15
minutes, rinsed under running tap water for 5 minutes, and air
dried. Counterstaining was performed with a 1:10 dilution of
Richardson stain at 45uC for 30 seconds. Sections were washed in
Figure 5. Co-administration of AT2220 promotes greater tissue glycogen reduction in GAA KO mice. Twelve-week old male GAA KO
mice were administered vehicle (water) or AT2220 (10 or 30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each
AT2220 administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 21 days after the
last (i.e.,4
th
) rhGAA administration and tissue glycogen levels were measured as described in ‘Materials and Methods’. Dotted lines show glycogen
levels in the respective tissues of wild-type C57BL/6 mice. The data presented are an average of two independent studies with each bar representing
the mean6SEM of the activity measured from 12 mice per group. Statistically significant reductions were seen in glycogen levels compared to
baseline (*p,0.05, t-test) and compared to rhGAA administration alone (#p,0.05, t-test). In addition, the effect of AT2220 co-administration was also
found to be significant for a linear trend (p,0.05; except in triceps), indicating a dose-dependent effect.
doi:10.1371/journal.pone.0040776.g005
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40776

deionized water, air dried, and mounted in Acrytol (Surgipath
Medical Industries, Richmond IL).
Quadriceps were fixed in 3% glutaraldehyde for 48 hours at
4uC and post-fixed in 1% periodic acid/neutral-buffered formalin
(48 hours, 4uC) before processing and paraffin embedding. Tissues
were sectioned at 5 mm on an HM 325 microtome (MICROM
International, GmbH, Walldorf, Germany). Sections were then
dewaxed in xylene and treated with dimedone to reduce
background staining during the subsequent PAS reaction. Finally,
sections were counterstained in Mayer’s hematoxylin, cleared in
xylene, and mounted in Acrytol (Surgipath Medical Industries).
Data Analysis
Determinations of statistical significance were conducted using
Excel 2003 (Microsoft, Redmond, WA) or GraphPad Prism,
version 5 (San Diego, CA) as defined in the figure and table
legends. Linear trends for dose-dependence were calculated using
a one-way ANOVA in GraphPad Prism. The half-life of rhGAA in
plasma was calculated using a non-linear one-phase exponential
decay curve fitting function in GraphPad Prism.
Results
AT2220 Stabilizes rhGAA, Preventing Denaturation and
Loss of Activity
The effect of AT2220 binding on the stability of rhGAA was
assessed using a fluorescence-based denaturation assay described
previously [28]. At 37uC, rhGAA was significantly less stable at
neutral pH than at acidic pH, with increases in SYPRO Orange
fluorescence occurring over 24 hours (Fig. 1A). Importantly, co-
incubation with 50 mM AT2220 at neutral pH significantly
stabilized rhGAA and prevented denaturation for up to 24 hours,
similar to observations made with the apo-enzyme at acidic pH
(Fig. 1A). Furthermore, incubation of rhGAA at neutral pH/37uC
resulted in a time-dependent loss of GAA activity, with a half-life of
3 to 4 hours (Fig. 1B). In contrast, incubation in acidic buffer, or
in neutral pH buffer containing 50 mM AT2220, prevented loss of
rhGAA activity for up to 24 hours (Fig. 1B). Similarly, incubation
of rhGAA in human whole blood resulted in a time-dependent loss
of activity; co-incubation with 50 mM AT2220 stabilized the
enzyme and prevented loss of activity for up to 24 hours (Fig. 1C).
Taken together, these results demonstrate that AT2220 stabilizes
rhGAA, preventing pH-, temperature-, and time-dependent
denaturation and inactivation.
AT2220 Co-administration Increases the Circulating Half-
life of rhGAA in Rats
The effect of AT2220 co-administration on the stability of
rhGAA in vivo was assessed in 8-week old Sprague Dawley rats. A
single oral administration of either 3 or 30 mg/kg AT2220 (which
yield C
max
values of approximately 4 and 40 mM, and are
comparable to the C
max
values seen in humans following oral
administration of 50 and 600 mg, respectively [29]), was given 30
minutes prior to bolus intravenous administration of rhGAA to
maximize the physical interaction of the two in the circulation (i.e.,
at AT2220 T
max
). In the absence of AT2220, rhGAA activity
declined rapidly in plasma, with a half-life of approximately 1.4
hours (Fig. 2A, upper panel). In contrast, oral administration of
Figure 6. Co-administration of AT2220 promotes cell type-specific reduction of glycogen in GAA KO mice. Twelve-week old male GAA
KO mice were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each
AT2220 oral administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 21 days after
the last (i.e., 4
th
) rhGAA administration and glycogen levels in heart and quadriceps were measured immunohistochemically as described in ‘Materials
and Methods’. A strong glycogen signal, represented as dark blue or pink spots (denoted with arrows) in heart and quadriceps, respectively, was
observed. (*) indicates glycogen reduction in individual skeletal muscle fibers of quadriceps. The data shown are representative photomicrographs
from 6 mice/group (magnification: 20X).
doi:10.1371/journal.pone.0040776.g006
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40776

3 or 30 mg/kg AT2220 prior to rhGAA resulted in a dose-
dependent increase in the circulating half-life of rhGAA to 2.1 and
3.0 hours, respectively (Fig. 2A, upper panel). Western blotting
of select time points indicated that AT2220 oral pre-administra-
tion leads to greater rhGAA protein levels in plasma compared to
rhGAA administration alone. Co-administration of AT2220
increased the quantity of the 110 kDa GAA protein band, with
the effect being more pronounced at the 8- and 24-hour time
points. Importantly, rhGAA protein was undetectable 24 hours
after administration of rhGAA alone, whereas AT2220 pre-
administration led to a substantial increase in circulating rhGAA
protein at this time point (Fig. 2A, lower panel).
To more closely mimic the clinical setting, the effect of oral
administration of AT2220 prior to rhGAA was also investigated in
rats using a 60-minute intravenous infusion of rhGAA. In the absence
of AT2220, rhGAA showed a half-life of approximately 1.1 hours
(Fig. 2B, upper panel). Oral administration of 3 or 30 mg/kg
AT1001 30 minutes prior to the rhGAA infusion resulted in dose-
dependent and significant increases of 1.4- and 2.0-fold, respec-
tively, in the circulating half-life of rhGAA (Fig. 2B, upper
panel). A similar effect was seen on rhGAA protein levels
following oral pre-administration of AT2220, with the greatest
effects seen during the elimination phase (Fig. 2B, lower panel).
The effect of oral administration of AT2220 on rhGAA was also
investigated in GAA KO mice. Twelve-week old male GAA KO
mice were pre-administered AT2220 (30 mg/kg) every other week
for 8 weeks. Thirty minutes after each AT2220 oral administra-
tion, rhGAA (20 mg/kg) was administered via bolus tail vein
injection. Plasma samples were collected 0, 1, and 4 hours after the
last (i.e., 4
th
) rhGAA administration for measurement of GAA
activity. Similar to the observations in rats, AT2220 co-adminis-
tration to GAA KO mice led to increases in plasma rhGAA activity
that were 2.5- and 2.0-fold greater than those seen following
administration of rhGAA alone at the 1 and 4 hour time points,
respectively (Fig. 3, upper panel). Concomitantly, an increase in
rhGAA protein levels was also seen in plasma (Fig. 3, lower
panel). Collectively, these data indicate that AT2220 co-
administration increases the circulating levels of rhGAA in rats
and in GAA KO mice.
Co-administration of AT2220 Increases the Tissue Uptake
of rhGAA in GAA KO Mice
Preliminary studies demonstrated that a single bolus tail vein
injection of rhGAA results in a dose-dependent increase in GAA
activity in disease-relevant tissues of GAA KO mice 7 days post-
administration, with 40 mg/kg rhGAA showing the greatest
effects (Fig. S1). Subsequently, repeat-administration studies were
conducted in GAA KO mice in the absence and presence of
30 mg/kg AT2220, a dose that yields a C
max
of ,40 mM, similar
to that seen in humans following oral administration of 600 mg
AT2220 [29]. Twelve-week old male GAA KO mice were
administered either vehicle (water) or AT2220 via oral gavage
once every other week for 8 weeks. Thirty minutes later, vehicle
(saline) or rhGAA (20 mg/kg; the recommended clinical dose) was
administered via bolus tail vein injection. Mice were euthanized 7
days after the final (4
th
) administration of rhGAA and tissue GAA
levels were measured. Oral pre-administration of AT2220 resulted
in increased GAA activity that was 2.5-, 2.2-, 1.5-, 1.7-, 1.7-, and
2.0-fold greater in heart, diaphragm, gastrocnemius, quadriceps,
triceps, and tongue, respectively, compared to administration of
rhGAA alone (Fig. 4).
Co-administration of AT2220 Leads to Greater Tissue
Glycogen Reduction in GAA KO Mice
Next, we determined the effect of AT2220 co-administration on
rhGAA-mediated glycogen reduction. Twelve-week old male GAA
KO mice were pre-administered either vehicle (water) or AT2220
(10 or 30 mg/kg) via oral gavage once every other week for 8
weeks. Thirty minutes after each oral administration of AT2220,
vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus
tail vein injection. Mice were euthanized 21 days after the final
(4
th
) administration of rhGAA and glycogen levels were measured.
Co-administration resulted in significantly greater tissue glycogen
reduction compared to rhGAA administration alone that was
generally dose-dependent (Fig. 5). Importantly, co-administration
with AT2220 resulted in maximally 1.6-, 1.3-, 2.6-, 2.0-, 2.4-, and
1.4-fold greater glycogen reduction in heart, diaphragm, quadri-
ceps, gastrocnemius, triceps, and tongue, respectively, compared
to rhGAA administration alone (Table 2). Notably, 20 mg/kg
rhGAA co-administered with 30 mg/kg AT2220 resulted in
glycogen reductions that were comparable to or greater than
those reported previously following administration of 40 mg/kg
rhGAA alone [32]. Furthermore, immunohistochemical measure-
ments confirmed that co-administration of 30 mg/kg AT2220
with 20 mg/kg rhGAA results in greater substrate reduction as
detected by reduced glycogen in individual smooth muscle and
skeletal muscle fibers of the heart and quadriceps, respectively
(Fig. 6).
Discussion
Regular infusion of rhGAA is currently the primary treatment
for Pompe disease. However, rhGAA has some limitations
including a short circulating half-life [33], inefficient uptake into
key tissues [14–15,34], and generation of immune responses that
can affect tolerability and efficacy [17]. In addition to the above
limitations, our prior [22] and current studies demonstrate that at
Table 2. Effect of AT2220 co-administration on rhGAA-
mediated glycogen reduction (% change from Baseline) in
GAA KO mice.
Tissues
2AT2220
+AT2220
(10 mg/kg) +AT2220 (30 mg/kg)
Heart 25465* 26865* 28562*
#
Diaphragm 24866* 24965* 26066*
#
Quadriceps 22064* 24563*
#
25264*
#
Gastrocnemius 2216724164* 24565*
#
Triceps 2106423363*
#
22463*
#
Tongue 24463* 25466*
#
26264*
#
Twelve-week old male GAA KO mice were administered vehicle (water) or
AT2220 via oral gavage once every other week for 8 weeks. Thirty minutes after
each AT2220 oral administration, rhGAA (20 mg/kg) was administered via bolus
tail vein injection. Mice were euthanized 21 days after the last (i.e., 4
th
) rhGAA
administration, and tissue glycogen levels were measured as described in
‘Materials and Methods’. Baseline glycogen levels in untreated GAA KO mice
were 417632, 340624, 405627, 353621, 446618, and 389610 mg/mg protein
in heart, diaphragm, quadriceps, gastrocnemius, triceps, and tongue,
respectively, and in wild-type C57BL/6 mice were 2362, 5066, 4065, 4562,
3567, and 3262mg/mg protein, respectively (mean6SEM of 7 mice). The data
shown represent the percent glycogen change from baseline in each tissue as
normalized between wild-type (0%) and GAA KO (100%) levels. Each value
represents the mean6SEM of 12 mice. Statistically significant reductions were
seen in glycogen levels compared to baseline (*p,0.05, t-test) and compared
to rhGAA administration alone (#p,0.05, t-test).
doi:10.1371/journal.pone.0040776.t002
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40776

body temperature, rhGAA is significantly less stable at neutral pH
than at acidic pH, which can lead to rapid loss of activity. This
instability at neutral pH is noteworthy, but not surprising given
that the endogenous enzyme is a resident lysosomal hydrolase with
a reported lysosomal half-life of ,10 days [35]. AT2220 is a PC
that has been shown to selectively bind endogenous GAA,
increasing its physical stability, lysosomal trafficking, and activity
in cultured cells [21–22]. Our current studies indicate that the
binding of AT2220 to exogenous rhGAA significantly increases the
physical stability and prevents denaturation of the enzyme at 37
uC in neutral pH buffer in vitro and in human blood ex vivo. The
binding and stabilization of rhGAA by a small molecule PC may
explain the increase in rhGAA cellular levels and tissue uptake that
were reported in a previous study that utilized the AT2220
derivative N-butyl-DNJ in combination with rhGAA [24].
Recently, PCs have also been shown to increase the stability and
to improve the cellular and tissue uptake of two other exogenous
recombinant enzymes that are used to treat LSDs, namely
recombinant human acid b-glucosidase [27] and a-galactosidase
A [24–26]. In the case of a-galactosidase A, greater substrate
reduction was also realized when used in combination with a PC
[25–26].
Similar to the stabilizing effects seen in blood ex vivo, AT2220
co-administration to mice and rats also prolonged the half-life of
rhGAA in the circulation. Importantly, these effects were seen at
doses (3 and 30 mg/kg) that result in plasma C
max
concentra-
tions of approximately 4 and 40 mM in rodents, which are
comparable to those achieved in humans following oral
administration of 50 and 600 mg AT2220, respectively [29].
Furthermore, AT2220 co-administration also resulted in up to
2.5-fold higher levels of GAA activity in disease-relevant tissues
of GAA KO mice compared to administration of rhGAA alone.
We hypothesize that the binding of AT2220 to rhGAA may
sufficiently increase the physical stability of the exogenous
enzyme to minimize or prevent thermally- and neutral pH-
mediated denaturation, as well as proteolysis, in the blood. A
longer circulating half-life of the stabilized, properly folded,
functional enzyme may increase the likelihood for recognition by
cation-independent mannose 6-phosphate receptors (CI-MPRs)
and subsequent uptake into disease-relevant cells and tissues
leading to improved substrate reduction. In addition, AT2220-
mediated stabilization of rhGAA in lysosomes, vesicles, and other
non-lysosomal compartments that are involved in the endocytic
pathway could lead to less or slower intracellular degradation of
rhGAA, and hence higher cellular/tissue levels. Importantly, co-
localization studies with fluorescent rhGAA and LAMP2 have
shown that PCs can improve the delivery of both endogenous
and exogenous GAA to lysosomes, resulting in higher cellular
levels of the fully-processed, mature forms of GAA [22,24].
These mature, lysosomal forms with molecular weights of 70 and
76 kDa more effectively catalyze glycogen turnover compared to
the 110 kDa precursor form [36–37].
Notably, co-administration of AT2220 and rhGAA lead to
greater glycogen reduction in tissues of GAA KO mice. Whereas
four injections of 20 mg/kg rhGAA showed little effect on the
glycogen levels of some skeletal muscles such as gastrocnemius and
triceps, co-administration with AT2220 resulted in significant
reductions. Furthermore, co-administration of 20 mg/kg rhGAA
with AT2220 resulted in glycogen reductions that were similar to
those reported previously with 40 mg/kg rhGAA alone [32],
suggesting that AT2220 can improve the potency of rhGAA.
While glycogen reduction was significantly greater following co-
administration of AT2220 and rhGAA, complete correction has
yet to be achieved (i.e., tissue glycogen levels do not reach the levels
seen in wild-type mice). This may be due to a number of factors.
First, wild-type tissue GAA levels are not realized even upon co-
administration of rhGAA and AT2220 at the doses tested. In fact,
total GAA tissue levels achieved with co-administration of AT2220
were maximally 30% to 60% of wild-type levels for a limited
amount of time. Second, it has been shown that rhGAA is often
mistrafficked in Pompe cells due to abnormal recycling of the CI-
MPR [15–16], which is essential for rhGAA uptake and delivery to
late endosomal/lysosomal compartments. Hence, while more
rhGAA is taken up into cells and tissues, and higher lysosomal
levels are achieved, it is possible that some of the exogenous
enzyme is delivered to inappropriate cellular compartments (i.e.,
not lysosomes), and hence is not accessible to some of the glycogen
pools [16]. Third, current commercial preparations of rhGAA
have poor affinity for the CI-MPR due to low mannose 6-
phosphate (Man6-P) content [32,38]. This, combined with a
reduced abundance of the CI-MPR in skeletal muscles of Pompe
mice [39], may limit delivery of rhGAA to lysosomes. Currently,
we do not know if AT2220 can increase the affinity of rhGAA for
the CI-MPR. However, a new form of rhGAA that is conjugated
to synthetic oligosaccharides that carry high levels of Man6-P
(oxime-neo-rhGAA) showed increased affinity for the CI-MPR,
and approximately 5-fold greater efficacy for reducing glycogen
compared to the unmodified form [38]. Evaluation of this
chemically-modified form of rhGAA, or other forms with modified
targeted motifs (NCT01435772; NCT01230801), in combination
with AT2220 is warranted to determine if increased physical
stability of high-affinity rhGAA may further improve tissue uptake
and glycogen reduction in GAA KO mice. Lastly, rhGAA is highly
immunogenic, which may impact its activity in vivo.
To this end, repeat administrations of rhGAA to GAA KO mice
leads to the development of a severe immune response due to the
development of anti-GAA antibodies. Due to the severity of the
immune response in these mice, the duration of the studies are
limited to four rhGAA administrations or less (due to mortalities
that arise beginning with the third rhGAA administration), again
potentially affecting the maximum long-term efficacy that can be
realized in a preclinical setting. An established line of immune-
tolerant mice [40] or a recently developed transgenic mouse model
that expresses low levels of a PC-responsive mutant form of human
GAA (P545L) on a GAA KO background [41] can be used for
future long-term rhGAA efficacy studies in combination with
AT2220.
Similar to the observations in mice, the cross-reactive immu-
nologic material (CRIM) status of Pompe patients can substantially
influence the efficacy and/or tolerability of ERT in the clinic [17].
Pompe patients who do not produce native enzyme (referred to as
CRIM-negative) are more prone to develop a sustained immune
response with high anti-GAA antibody titers (some of which can
be neutralizing), compared to CRIM-positive patients [18].
Treatment of patients with immunomodulatory agents such as
methotrexate has led to improved ERT-treatment outcomes in
CRIM-negative Pompe patients [42] and in mice [43]; similarly,
genetically- or chemically-induced immune tolerance has been
shown to significantly reduce IgG levels in GAA KO mice [44–46].
Furthermore, misfolded, denatured, and/or aggregated therapeu-
tic proteins are known to be more immunogenic than correctly
folded, stable protein therapeutics [47]. Thus, it is possible that the
ERT-mediated immunogenicity observed in many Pompe patients
results from destabilization or denaturation of rhGAA in the blood
(and possibly in the infusion solution). By stabilizing rhGAA in its
correctly folded, monomeric, native conformation, we hypothesize
that AT2220 co-administration may attenuate rhGAA-mediated
immunogenicity. Future repeat-administration studies focused on
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40776

measuring IgG levels with rhGAA in combination with AT2220 in
GAA KO mice will be needed to further investigate these
possibilities.
Recent studies indicate that there is also a neuropathological
component to Pompe disease that is characterized by widespread
CNS pathology [48–50]. However, motoneurons seem particular-
ly susceptible to glycogen accumulation in both patients and GAA
KO mice, and reduced motor output has been described [48–50].
Though AT2220-mediated increases in ERT penetration into the
central nervous system are unlikely, it is possible that co-
administration could lead to greater rhGAA uptake into neurons
of the peripheral nervous system (e.g., phrenic nerves and sensory
ganglia) [48], potentially leading to greater glycogen reduction and
improved neuronal function. Future studies will be necessary to
evaluate this potential, particularly on phrenic nerve pathology
and overall respiratory function.
Collectively, our data indicate that AT2220 increases the
physical stability of rhGAA, and leads to higher cellular and tissue
activity as evidenced by greater substrate turnover in situ. As such,
co-administration may provide an improved therapeutic strategy
for the treatment of Pompe disease. Based on these encouraging
findings, a Phase 2 clinical study has been initiated to investigate
the combination of AT2220 with rhGAA in Pompe patients.
Supporting Information
Figure S1 Dose-dependent increases in tissue GAA
activity following administration of rhGAA to GAA KO
mice. Twelve-week old male GAA KO mice were administered
10, 20, or 40 mg/kg rhGAA via bolus tail vein injection. Mice
were euthanized 7 days later, and GAA activity was measured in
disease-relevant tissues (see ‘‘Materials and Methods’’ in the
original article). Administration of rhGAA led to dose-dependent
and significant increases (*p,0.05 compared to baseline, via t-test)
in tissue GAA levels, with the greatest activity seen with the
40 mg/kg dose. Each bar represents the mean6SEM for 4–7 mice
per group.
(TIF)
Acknowledgments
The authors wish to thank Dr. Barry Byrne and Nina Raben for creating
the original GAA KO mice, and Dr. Hung Do for initiating and
supervising the in vitro stability assays. Thanks also to Eurofins Product
Safety Labs (Dayton, NJ) for conducting the in-life portion of the study
highlighted in Fig. 2A, ITR Inc. (Montreal, Canada) for the study
highlighted in Fig. 2B, and the EM Core Labs (Department of
Molecular Biology, Princeton University, Princeton, NJ) for processing
the heart tissues presented in Fig. 6.
Author Contributions
Conceived and designed the experiments: RK JJF DJL KJV. Performed
the experiments: RK JJF JF RS MF LJP YL DG. Analyzed the data: RK
JJF KJV. Wrote the paper: RK KJV.
References
1. Hirschhorn R, Reuser AJJ (2001) Glycogen storage disease type ii: Acid alpha-
glucosidase (acid maltase) deficiency. In: Scriver C, Beaudet A, Sly W, Valle D,
editors. The metabolic and molecular bases of inherited disease. New York:
McGraw-Hill. 3389–3420.
2. van der Ploeg AT, Reuser AJJ (2008) Pompe’s disease. Lancet 372: 1342–1353.
3. van den Hout HM, Hop W, van Diggelen OP, Smeitink JA, Smit GP, et al.
(2003) The natural course of infantile pompe’s disease: 20 original cases
compared with 133 cases from the literature. Pediatrics 112: 332–340.
4. Kishnani PS, Howell RR (2004) Pompe disease in infants and children. J Pediatr
144: S35–43.
5. Kishnani PS, Nicolino M, Voit T, Rogers RC, Tsai AC, et al. (2006) Chinese
hamster ovary cell-derived recombinant human acid alpha-glucosidase in
infantile-onset pompe disease. J Pediatr 149: 89–97.
6. Van den Hout H, Reuser AJ, Vulto AG, Loonen MC, Cromme -Dijkhuis A, et
al. (2000) Recombinant human alpha-glucosidase from rabbit milk in pompe
patients. Lancet 356: 397–398.
7. Van den Hout JM, Kamphoven JH, Winkel LP, Arts WF, De Klerk JB, et al.
(2004) Long-term intravenous treatment of pompe disease with recombin ant
human alpha-glucosidase from milk. Pediatrics 113: e448–457.
8. Nicolino M, Byrne B, Wraith JE, Leslie N, Mandel H, et al. (2009) Clinical
outcomes after long-term treatment with alglucosidase alfa in infants and
children with advanced pompe disease. Genet Med 11: 210–219.
9. Chen L-R, Chen C-A, Chiu S-N, Chien Y-H, Lee N-C, et al. (2009) Reversal of
cardiac dysfunction after enzyme replacement in patients with infantile-onset
pompe disease. J Pediatr 155: 271–275.
10. Strothotte S, Strigl-Pill N, Grunert B, Kornblum C, Eger K, et al. (2010)
Enzyme replacement therapy with alglucosidase alfa in 44 patients with late-
onset glycogen storage disease type 2: 12-month results of an observational
clinical trial. J Neurol 257: 91–97.
11. van der Ploeg AT, Clemens PR, Corzo D, Escolar DM, Florence J, et al. (2010)
A randomized study of alglucosidas e alfa in late-onset pompe’s disease.
N Engl J Med 362: 1396–1406.
12. Thurberg BL, Lynch Maloney C, Vaccaro C, Afonso K, Tsai AC-H, et al.
(2006) Characterization of pre- and post-treatment pathology after enzyme
replacement therapy for pompe disease. Lab Invest 86: 1208–1220.
13. Schoser B, Hill V, Raben N (2008) Therapeutic approaches in glycogen storage
disease type ii/pompe disease. Neurotherapeutics 5: 569–578.
14. Raben N, Danon M, Gilbert AL, Dwivedi S, Collins B, et al. (2003) Enzyme
replacement therapy in the mouse model of pompe disease. Mol Genet Metab
80: 159–169.
15. Fukuda T, Roberts A, Ahearn M, Zaal K, Ralston E, et al. (2006) Autophagy
and lysosomes in pompe disease. Autophagy 2: 318–320.
16. Cardone M, Porto C, Tarallo A, Vicinanza M, Rossi B, et al. (2008) Abnormal
mannose-6-phosphate receptor trafficking impairs recombinant alpha-glucosi-
dase uptake in pompe disease fibroblasts. Pathogenetics 1: 6.
17. Kishnani PS, Goldenberg PC, DeArmey SL, Heller J, Benjamin D, et al. (2010)
Cross-reactive immunologic material status affects treatment outcomes in pompe
disease infants. Mol Genet Metab 99: 26–33.
18. de Vries JM, van der Beek NAME, Kroos MA, O
¨zkan L, van Doorn PA, et al.
(2010) High antibody titer in an adult with Pompe disease affects treatment with
alglucosidase alfa. Mol Genet Metab 101: 338–345.
19. Fan J-Q (2008) A counterintuitive approach to treat enzyme deficiencies: Use of
enzyme inhibitors for restoring mutant enzyme activity. Biol Chem 389: 1–11.
20. Okumiya T, Kroos MA, Vliet LV, Takeuchi H, Van der Ploeg AT, et al. (2007)
Chemical chaperones improve transport and enhance stability of mutant alpha-
glucosidases in glycogen storage disease type ii. Mol Genet Metab 90: 49–57.
21. Parenti G, Zuppaldi A, Gabriela Pittis M, Rosaria Tuzzi M, Annunziata I, et al.
(2007) Pharmacological enhancement of mutated alpha-glucosidase activity in
fibroblasts from patients with pompe disease. Mol Ther 15: 508–514.
22. Flanagan JJ, Rossi B, Tang K, Wu X, Mascioli K, et al. (2009) The
pharmacological chaperon e 1-deoxynojirimycin increases the activity and
lysosomal trafficking of multiple mutant forms of acid alpha-glucosidase. Hum
Mutat 30: 1683–1692.
23. Valenzano KJ, Khanna R, Powe AC, Boyd R, Lee G, et al. (2011) Identification
and characterization of pharmacological chaperones to correct enzyme
deficiencies in lysosomal storage disorders. Assay Drug Dev Technol 9: 213–235.
24. Porto C, Cardone M, Fontana F, Rossi B, Tuzzi MR, et al. (2009) The
pharmacological chaperone n-butyldeoxynojirimycin enhances enzyme replace-
ment therapy in pompe disease fibroblasts. Mol Ther 17: 964–971.
25. Porto C, Pisani A, Rosa M, Acampora E, Avolio V, et al. (2012) Synergy
between the pharmacological chaperone 1-deoxygalactonojirimycin and the
human recombinant alpha-galactosidase a in cultured fibroblasts from patients
with fabry disease. J Inherit Metab Dis 35(3): 513–520.
26. Benjamin ER, Khanna R, Schilling A, Flanagan JJ, Pellegrino LJ, et al. (2012)
Co-administration with the pharmacological chaperone at1001 increases
recombinant human alpha-galactosidase a tissue uptake and improves substrate
reduction in fabry mice. Mol Ther: 20: 717–726.
27. Shen J-S, Edwards NJ, Hong YB, Murray GJ (2008) Isofagomine increa ses
lysosomal delivery of exogenous glucocerebrosidase. Biochem Biophys Res
Commun 369: 1071–1075.
28. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning
fluorimetry to detect ligand interactions that promote protein stability. Nat
Protocols 2: 2212–2221.
29. Adera M, Boudes P, Bragat A, Sitaraman S, Lazauskas R, et al. (2011)
Pharmacokinetics and muscle distribution of at2220, a pharmacological
chaperone of acid-glucosidase, in healthy volunteers. Mol Genet Metab 102: S4.
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e40776

30. Amalfitano A, McVie-Wylie AJ, Hu H, Dawson TL, Raben N, et al. (1999)
Systemic correction of the muscle disorder glycogen storage disease type ii after
hepatic targeting of a modified adenovirus vector encoding human acid-alpha-
glucosidase. Proc Natl Acad Sci USA 96: 8861–8866.
31. Lynch CM, Johnson J, Vaccaro C, Thurberg BL (2005) High-resolution light
microscopy (hrlm) and digital analysis of pompe disease pathology. J Histochem
Cytochem 53: 63–73.
32. Zhu Y, Li X, McVie-Wylie A, Jiang C, Thurberg BL , et al. (2005)
Carbohydrate-remodelled acid alpha-glucosidase with higher affinity for the
cation-independent mannose 6-phosphate receptor demonstrates improved
delivery to muscles of pompe mice. Biochem J 389: 619–628.
33. Myozyme package insert (2006) Alglucosidase alfa. Cambridge, MA: Genzyme
Corp. Available: http://www.myozyme.com/˜
/media/Files/MyozymeUS/
Documents/mz_pi.pdf. Accessed 2012 Jun 19.
34. Drost MR, Schaart G, Dijk Pv, Capelle CIv, Vusse GJvD, et al. (2008) Both type
1 and type 2a muscle fibers can respond to enzyme therapy in pompe disease.
Muscle Nerve 37: 251–255.
35. Reuser AJJ, Kroos MA, Ponne NJ, Wolterman RA, Loonen MCB, et al. (1984)
Uptake and stability of human and bovine acid [alpha]-glucosidase in cultured
fibroblasts and skeletal muscle cells from glycogenosis type ii patients. Exp Cell
Res 155: 178–189.
36. Wisselaar HA, Kroos MA, Hermans MM, van Beeumen J, Reuser AJ (1993)
Structural and functional changes of lysosomal acid alpha-glucosidase during
intracellular transport and maturation. J Biol Chem 268: 2223–2231.
37. Bijvoet AG, van de Kamp EH, Kroos MA, Ding JH, Yang BZ, et al. (1998)
Generalized glycogen storage and cardiomegaly in a knockout mouse model of
pompe disease. Hum Mol Genet 7: 53–62.
38. Zhu Y, Jiang J-L, Gumlaw NK, Zhang J, Bercury SD, et al. (2009)
Glycoengineered acid alpha-glucosidase with improved efficacy at correcting
the metabolic aberrations and motor function deficits in a mouse model of
pompe disease. Mol Ther 17: 954–963.
39. Koeberl DD, Luo X, Sun B, McVie-Wylie A, Dai J, et al. (2011) Enhanced
efficacy of enzyme replacement therapy in pompe disease through mannose-6-
phosphate receptor expression in skeletal muscle. Mol Genet Metab 103: 107–
112.
40. Raben N, Nagaraju K, Lee A, Lu N, Rivera Y, et al. (2003) Induction of
tolerance to a recombinant human enzyme, acid alpha-glucosidase, in enzyme
deficient knockout mice. Transgenic Res 12: 171–178.
41. Khanna R, Soksa R, Feng J, Lun Y, Powe AC, et al. (2009) G.P.8.05 the
pharmacological chaperone at2220 increases mutant acid alpha-glucosidase
levels and reduces tissue glycogen in a mouse model of pompe disease.
Neuromuscul Disord 19: 592.
42. Mendelsohn NJ, Messinger YH, Rosenberg AS, Kishnani PS (2009) Elimination
of antibodies to recombinant enzyme in pompe’s disease. N Engl J Med 360:
194–195.
43. Sun B, Kulis MD, Young SP, Hobeika AC, Li S, et al. (2009) Immunomod-
ulatory gene therapy prevents antibody formation and lethal hypersensitivity
reactions in murine pompe disease. Mol Ther 18: 353–360.
44. Joseph A, Munroe K, Housman M, Garman R, Richards S (2008) Immune
tolerance induction to enzyme-replacement therapy by co-administration of
short-term, low-dose methotrexate in a murine pompe disease model. Clin Exp
Immunol 152: 138–146.
45. Douillard-Guilloux G, Mouly V, Caillaud C, Richard E (2009) Immortalization
of murine muscle cells from lysosomal alpha-glucosidase deficient mice: A new
tool to study pathophysiology and assess therapeutic strategies for pompe
disease. Biochem Biophys Res Commun 388: 333–338.
46. van Til NP, Stok M, Aerts Kaya FSF, de Waard MC, Farahbakhshi an E, et al.
(2010) Lentiviral gene therapy of murine hematopoietic stem cells ameliorates
the pompe disease phenotype. Blood 115: 5329–5337.
47. Thai R, Moine G, Desmadril M, Servent D, Tarride J-L, et al. (2004) Antigen
stability controls antigen presentation. J Biol Chem 279: 50257–50266.
48. DeRuisseau LR, Fuller DD, Qiu K, DeRuisseau KC, Donnelly WH, et al.
(2009) Neural deficits contribute to respiratory insufficiency in pompe disease.
Proceedings of the National Academy of Sciences 106: 9419–9424.
49. Sidman RL, Taksir T, Fidler J, Zhao M, Dodge JC, et al. (2008) Temporal
neuropathologic and behavioral phenotype of 6neo/6neo pompe disease mice.
J Neuropathol Exp Neurol 67: 803–818.
50. Lee C-J, Fan X, Guo X, Medin JA (2011) Promoter-specific lentivectors for
long-term, cardiac-directed therapy of fabry disease. J Cardiol 57: 115–122.
Effect of AT2220 Co-Administration on rhGAA
PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e40776