original article
Evaluation of Systemic Follistatin as an Adjuvant
to Stimulate Muscle Repair and Improve
Motor Function in Pompe Mice
Joseph W Foley1, Scott D Bercury1, Patrick Finn1, Seng H Cheng1, Ronald K Scheule1 and Robin J Ziegler1
Genzyme Corporation, Framingham, Massachusetts, USA
1
Due to the lack of acid α-glucosidase (GAA) activity,
Pompe mice develop glycogen storage pathology and
progressive skeletal muscle dysfunction with age. Apply-
ing either gene or enzyme therapy to reconstitute GAA
levels in older, symptomatic Pompe mice effectively
reduces glycogen storage in skeletal muscle but pro-
vides only modest improvements in motor function.
As strategies to stimulate muscle hypertrophy, such as
by myostatin inhibition, have been shown to improve
muscle pathology and strength in mouse models of
muscular dystrophy, we sought to determine whether
these ­benefits might be similarly realized in Pompe mice.
Administration of a recombinant adeno-associated virus
serotype 8 vector encoding follistatin, an inhibitor of
myostatin, increased muscle mass and strength but only
in Pompe mice that were treated before 10 months of
age. Younger Pompe mice showed significant muscle
fiber hypertrophy in response to treatment with follista-
tin, but maximal gains in muscle strength were achieved
only when concomitant GAA administration reduced
glycogen storage in the affected muscles. Despite
increased grip strength, follistatin treatment failed to
improve rotarod performance. These findings high-
light the importance of treating Pompe skeletal muscle
before pathology becomes irreversible, and suggest that
adjunctive therapies may not be effective without first
clearing skeletal muscle glycogen storage with GAA.
Received 4 February 2010; accepted 12 May 2010; published online
15 June 2010. doi:10.1038/mt.2010.110
Introduction
Pompe disease, a lysosomal storage disorder, is characterized by
progressive degenerative myopathy. Deficiency of the lysosomal
enzyme acid α-glucosidase (GAA) results in massive accumula-
tion of glycogen in lysosomes and autophagosomes of striated
and smooth muscle, leading to skeletal muscle weakness and
cardiorespiratory failure.1,2 Patients present clinically with a spec-
trum of disease severity that inversely correlates with residual
enzyme activity.
A Pompe mouse model (6neo/6neo) has been described that
shows negligible enzyme activity and a pattern of glycogen storage
Correspondence: Robin J Ziegler, Genzyme Corporation, 49 New York Avenue, Framingham, Massachusetts, USA. E-mail: robin.ziegler@genzyme.com
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© The American Society of Gene & Cell Therapy
similar to human patients.3 Mice are healthy at birth and breed
normally, but demonstrate muscle pathology and weakness with
age. Detailed ultrastructural studies have drawn parallels between
the muscle pathology in the Pompe mouse and affected humans.4,5
We have shown previously that systemic delivery of an adeno-
associated virus serotype 8 (AAV8) vector encoding GAA in young,
presymptomatic Pompe mice reduced glycogen to basal levels and
prevented motor function loss.6 However, when this treatment was
applied to older, disease impaired Pompe mice, only partial motor
function improvement resulted. In an effort to improve functional
outcome in these older mice, we evaluated the potential of an adju-
vant, namely follistatin, to improve motor function.
Inhibiting myostatin, a negative regulator of muscle growth,
has been shown to increase muscle mass and improve function
in some mouse models of neuromuscular disorders.7–12 These
effects may be attributed, at least in part, to enhanced muscle
regeneration.13,14 Although various myostatin inhibitors have been
described,7–10,15–17 follistatin can modulate other regulators of mus-
cle mass in addition to myostatin. For example, follistatin admin-
istration to Mstn−/− mice caused muscle mass increases beyond
that stimulated by myostatin depletion18,19 suggesting that this
may be a more potent approach than targeting myostatin alone.
Intramuscular administration of gene therapy vectors expressing
follistatin have increased muscle mass and strength in both young
and aged mdx mice,11 as well as in nonhuman primates.20
To determine whether systemic follistatin could affect global
musculature and improve functional efficacy in aged Pompe mice,
we used liver-directed AAV8 vectors to provide the secreted form
of human follistatin.21,22 We administered AAV8-GAA to symp-
tomatic Pompe mice to clear glycogen from the muscles, and co-­
administered AAV8-follistatin to stimulate muscle regeneration
and/or hypertrophy. We chose liver-directed AAV8 as a delivery
vehicle to generate prolonged circulating levels of the therapeutic
proteins, and to prevent complications from neutralizing anti­
bodies to the expressed transgenes,6,23–27 thereby enabling us to
­perform long-term functional analyses of the treated Pompe mice.
Results
Pompe mice demonstrate progressive motor
function deficits with age
In previous studies, we had observed that as Pompe mice
aged, their muscle strength and coordination degenerated,
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© The American Society of Gene & Cell Therapy
Evaluation of Systemic Follistatin in Pompe Mice

a Rotarod b Grip strength (front) c Grip strength (all)

100 140 350
*** ^^ **
*
80 120 300
Latency (seconds)
^
^
250

Force (g)

Force (g)
60 100
200
40 80
150
20 60 100
0 40 50
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Months of age Months of age Months of age

B6.129 129S2 C57B6 Pompe

Figure 1  Pompe mice demonstrate motor function deficits. Pompe mice and age-matched wild-type mice were subjected to a series of assays to
evaluate motor function and muscle strength. Testing was performed once a month. (a) Accelerating rotarod measures muscle strength, coordina-
tion, and endurance. Pompe mice demonstrated significant deterioration in performance as they aged compared to the relatively stable performance
of the wild-type strains. (b) Pompe mice showed reduced front limb grip strength compared to 129S2 or B6.129 mice, but performance was similar
to C57B6 wild type. (c) Pompe mice showed significantly reduced force compared to all wild-type strains when grip strength testing included all
limbs. Data are expressed as average ± SEM (n = 10/group); asterisks denote *P < 0.05, **P < 0.01, ***P < 0.001, Pompe compared to all wild-type
strains; carets denote ∧P < 0.05, ∧∧P < 0.01, Pompe compared to 129S2 or B6.129 only.

as measured by rotarod and wire hang tests.6 Administration

of AAV8-GAA to asymptomatic 3-month-old Pompe mice a 10
Follistatin serum levels b 1.5 Lean mass c 300 Quad weight
** **
**
prevented this decline in motor function. However, treating *** 250
Follistatin (µg/ml)

Wet weight (mg)

1
**

Fold change
200
symptomatic 10-month-old Pompe mice did not normalize 0.1 1 150
their performance in these tests.6 To evaluate the potential of 100
0.01
follistatin to improve muscle strength in 10-month-old Pompe 50

mice, we applied less demanding functional tests in an attempt 0.001

0 1 2 3 4 5 6
0.5
0 1 2 3 4 5 6
0
Vehicle Follistatin
to discern more incremental changes in muscle strength, e.g., Weeks post-administration Weeks post-administration

by deploying a decreased rate of ­acceleration on the rotarod Vehicle Follistatin

test. Despite a slower rotarod acceleration rate, the pattern

of functional decline in the Pompe mice over time remained
unchanged, with latency declining to ~10 seconds by 17 months
of age (Figure  1a). In contrast, all three parental wild-type
strains maintained their initial latency of ~70 seconds under
the same conditions.
Performance on the rotarod requires endurance and coordi-
nation as well as muscle strength. For a more specific measure
of muscle strength, grip strength tests were performed. Grip
strength measurements showed that compared to wild-type
mice, Pompe mice were significantly impaired at an early age,
but in contrast to the rotarod results, did not decline drastically
with increasing age (Figure 1b,c). When the front limbs alone
were tested, there was a modest but significant difference in
strength between Pompe and 129S2 or B6.129 wild-type mice,
but no difference was observed between Pompe and C57B6 mice
(Figure 1b). Testing both front and rear limbs showed a greater
differential between Pompe and all the wild-type strains, with
the wild-type (but not Pompe) mice demonstrating increasing
strength over time (Figure 1c). These data support our observa-
tions that Pompe mice exhibit more functional deterioration in
the hind limbs.
Administration of AAV8-follistatin increased lean
and quadriceps mass in adult wild-type mice
The objective of these studies was to determine whether the con-
comitant delivery of follistatin and GAA could increase muscle
Figure 2  AAV8-Follistatin increased lean mass and quadriceps mass
in adult wild-type mice. Male C57B6 mice (3 months of age) were
injected systemically with AAV8-follistatin or vehicle and evaluated
over 5 weeks. (a) Serum was collected at the indicated time points and
human follistatin levels measured using an enzyme-linked immunosor-
bent assay. (b) Follistatin-treated mice showed a significant increase in
whole-body lean mass compared to vehicle-treated as determined by
EchoMRI. (c) At killing, the right quadriceps muscle was excised and
weighed. The quadriceps mass of the follistatin-treated mice was twice
that of the vehicle-treated controls. Data are expressed as average ± SEM
(n = 4–5/group); asterisks denote **P < 0.01, ***P < 0.001, follistatin
compared to vehicle.
mass and correct the functional deficits beyond that which
had been noted earlier with GAA alone in aged Pompe mice.
Before initiating long-term, labor-intensive functional studies
in Pompe mice, 1 × 1011 DNase resistant particles of AAV8-
follistatin or vehicle were initially administered into 3-month-
old wild-type (C57B6) mice to measure expression levels of
follistatin and its effects on muscle mass. Virus-treated mice
exhibited a sustained ­circulating level of ~0.4 µg/ml follistatin
(Figure  2a) that correlated with a significant increase (~15%)
in lean mass compared to vehicle-treated animals (Figure 2b).
After 9 weeks, mice treated with AAV8-follistatin showed a
doubling of the quadriceps mass over vehicle-treated mice (265
± 20 mg versus 126 ± 24 mg; Figure  2c). These data indicated
that sustained serum levels of ~0.4 µg/ml follistatin were suf-
ficient to stimulate significant skeletal muscle hypertrophy in
adult wild-type mice.

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Evaluation of Systemic Follistatin in Pompe Mice

a Glycogen b Lean mass c Grip strength (front) d Grip strength (all)

50 1.5 140 350
Vehicle
300
mg glycogen/g tissue

40 Follistatin 120
GAA * 250

Fold change
30

Force (g)

Force (g)
GAA and Follistatin 100
1 200
20 80
150
10 60 100
0 0.5 40 50
Heart Quad Triceps 8 10 12 14 16 13 14 15 16 13 14 15 16

e 50 f 1.5 g 140 h 350

^^ ^ ^
*** ** 300
mg glycogen/g tissue

40 120 *** ^
Fold change

250

Force (g)
Force (g)
30 100 **
1 200
20 80
150
10 60 100

0 0.5 40 50
Heart Quad Triceps 5 6 7 8 9 10 11 4 6 8 10 12 4 6 8 10 12
Tissue Months of age Months of age Months of age

Vehicle Follistatin GAA GAA and Follistatin

Figure 3 Systemic follistatin improved motor function in younger but not older GAA-treated Pompe mice. Ten- (top panels) or seven- (bottom
panels) month-old male Pompe mice were treated with AAV8-GAA, AVV8-Follistatin, a mixture of the GAA and follistatin vectors, or vehicle and were
killed ~5 months after virus administration. (a) AVV8-GAA treatment significantly reduced glycogen levels in muscles of mice treated at 10 months
of age. (b) Whole body lean mass, measured by Echo MRI, and (c,d) muscle strength, evaluated using grip strength assays, showed little change
regardless of treatment when vectors were administered at 10 months of age. (e) Muscles from animals treated at 7 months showed a similar extent
of glycogen clearance as in the older mice (40–60%). Mice treated at 7 months of age with GAA and follistatin showed significant increases in (f) lean
mass and (g,h) grip strength over those treated with GAA alone. Data are expressed as average ± SEM (n = 4–10/group); Asterisks denote *P < 0.05,
**P < 0.01, ***P < 0.001 GAA and follistatin compared to GAA alone; carets donote ∧P < 0.05, ∧∧P < 0.01, follistatin compared to vehicle. (P < 0.05
GAA & follistatin compared to follistatin alone for the last two time points in f and h).

Systemically delivered follistatin improved motor with or without GAA to these relatively old Pompe mice did not
function in younger but not older GAA-treated improve their grip strength (Figure 3c,d).
Pompe mice An analogous study was conducted in 7-month-old Pompe
Previously, we had reported that although enzyme augmenta- mice, an age at which they showed only a moderate decline in
tion therapy is able to reduce the lysosomal glycogen stores in rotarod function (Figure 1a). Treatment with the viral vectors gen-
the ­muscles of Pompe mice, this therapeutic strategy alone could erated similar serum levels of GAA and follistatin to those described
not completely reverse the accompanying myopathy, particu- above (data not shown). At 4–5 months post-treatment an analy-
larly in aged Pompe mice.6,28 To address this challenge, follista- sis of Pompe mice administered AAV8-GAA showed a 40–60%
tin was co-administered as an adjuvant with GAA to determine reduction in their skeletal muscle glycogen levels (Figure 3e). This
whether stimulating muscle regeneration and/or hypertrophy extent of glycogen clearance was comparable to that seen in the
could improve the functional response beyond that observed older mice (above). However, in contrast to the results with the
with GAA treatment alone. Pompe mice were treated beginning older Pompe mice, those that had been treated earlier (starting at
at 10  months, an age at which we have characterized extensive 7 months of age) with AAV8-follistatin demon­strated a significant
muscle pathology and dysfunction.6 Cohorts of Pompe mice increase in lean mass compared to those treated with AAV8-GAA
(n = 10) were administered AAV8-GAA, AAV8-follistatin, a mix- alone (Figure 3f). A significant increase in the mass of the quadri-
ture of both AAV8-GAA and AAV8-follistatin, or vehicle. GAA ceps muscles was also noted in the AAV8-follistatin-treated mice
and follistatin expression levels were similar across the different (Supplementary Figure S1). Associated with these gains in muscle
treatment groups, whether viral vectors were administered alone mass were significant improvements in grip strength (Figure 3g,h).
or in combination (~10 µg/­ml GAA and ~1 µg/ml follistatin; data The force generated when all limbs were tested in mice treated
not shown). As expected, the skeletal and heart muscles of mice with follistatin and GAA (180–200 g) was significantly greater than
treated with AAV8-GAA showed significant decreases in glycogen that produced in the vehicle- or GAA-treated mice (120–150 g).
levels (Figure 3a), similar to our previous results in Pompe mice However, treatment with GAA and follistatin did not increase grip
of this age.6 There was a trend toward an increase in lean mass strength near to the levels seen in the wild-type strains (225–305 g;
in animals administered AAV8-follistatin in combination with Figure  1). Importantly, the response to follistatin (as measured
AAV8-GAA (Figure  3b). However, the provision of follistatin by both muscle mass and grip strength) was greater in Pompe

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© The American Society of Gene & Cell Therapy
Evaluation of Systemic Follistatin in Pompe Mice

a b c
Treated at 10 months Treated at 7 months Treated at 5 months
60 60 60
* **

Latency (seconds)
50 50 50
40 40 40
30 30 30
20 20 20
10 10 10
0 0 0
8 10 12 14 16 4 6 8 10 12 2 4 6 8 10
Months of age Months of age Months of age
Vehicle Follistatin GAA GAA and Follistatin

Figure 4  Provision of follistatin did not improve rotarod ­performance in GAA-treated Pompe mice. Male Pompe mice were treated with GAA
and follistatin vectors (as previously described) at (a) 10 months, (b) 7 months, or (c) 5 months of age (as indicated by a black arrow), and tested
for endurance and coordination on a rotarod. The ­follistatin-treated mice showed no significant improvement in rotarod performance at any age. In
mice treated at 5 months of age, which had up to 90% clearance of glycogen in skeletal muscles, there was a trend for the mice treated with GAA
alone to perform best. Data are expressed as average ± SEM (n = 4–10/group); Asterisks denote *P < 0.05, **P < 0.01, GAA compared to follistatin
or vehicle.

a b a Average size – 7 months b Average size – 10 months

4,000 4,000
Fiber area (µm2) **
3,000 3,000 Vehicle
GAA
2,000 2,000 Follistatin
GAA and
1,000 1,000
Follistatin
0 0

c d c Size distribution – 7 months d Size distribution – 10 months

70 70
% Fibers per size group

60 60
50 50
40 40
30 30
20 20
10 10
0 0
0–1,000
1,001–2,000
2,001–3,000
3,001–4,000
4,001–5,000
5,001–6,500
6,501–8,000
8,001–10,000
>10,001

Figure 5  Histological evaluation of muscle fiber hypertrophy.
Representative sections of formalin-fixed, hematoxylin and eosin stained
quadriceps from the mice described in Figure  3. Photomicrographs
depict how muscle fibers were outlined (in black) and the cross-sectional
area measured using Aperio software. Qualitatively, in mice that had
been treated at 7 months of age, (a) vehicle-treated animals had fibers
with a smaller cross-sectional area than (b) mice treated with AAV8-GAA
and AAV8-follistatin. In mice treated at 10 months of age, this difference
between (c) vehicle and (d) AAV8-GAA plus AAV8-follistatin was less
apparent. There were some unusually large fibers found in the follistatin-
treated mice (white arrowheads). White areas within the cytoplasm likely
­represent vacuolar glycogen storage, or central cores of autophagic
debris. Bar = 100 µm.
mice that had also been treated with AAV8-GAA to decrease
muscle ­lysosomal glycogen stores (Figure  3f–h; Supplementary
Figure S1). In summary, Pompe mice that had been treated earlier
responded better to follistatin, suggesting that the muscle patho­
logy became more difficult to reverse with age. Follistatin and
GAA worked synergistically to increase muscle mass and improve
­muscle function in the younger Pompe mice.
Provision of follistatin did not improve rotarod
performance in GAA-treated Pompe mice
We had previously shown that treating young Pompe mice
(3 months of age) with AAV8-GAA preserved muscle endurance
0–1,000
1,001–2,000
2,001–3,000
3,001–4,000
4,001–5,000
5,001–6,500
6,501–8,000
8,001–10,000
>10,000
Fiber area (µm2) Fiber area (µm2)
Figure 6  Pompe mice demonstrated significant muscle fiber hyper-
trophy only when treated at 7 months of age. Muscle fiber hypertro-
phy was quantified from the slides described above. The area (µm2) of
each outlined muscle fiber was calculated for 250–500 fibers of 7–8 mice/
treatment group. (a) The average fiber size increased significantly in the
Pompe mice treated with AAV8-follistatin at 7 months of age, but (b) not
if treated at 10 months. (c,d) Fiber size distribution was also evaluated.
Pompe mice treated at 7 months of age with follistatin showed a shift
toward larger sized fibers (and fewer smaller sized fibers), with some that
were extremely large. Mice treated at 10 months of age showed only a
slight trend toward larger muscle fibers. Data are expressed as average ±
SEM (n = 7–8 per group); Asterisks denote **P < 0.01, follistatin treat-
ment groups compared to GAA alone or vehicle (there was no statistical
difference between mice treated with GAA and follistatin compared to
mice treated with follistatin alone).
and coordination as measured by rotarod, but treating older Pompe
mice (10 months of age) did not restore their ­ability to function in
this assay. The mice described in this study were similarly tested for
muscle endurance and coordination using the rotarod to determine
whether follistatin treatment could improve functional outcome.
The mice were treated with AAV8‑GAA and ­AAV8-follistatin

Molecular Therapy vol. 18 no. 9 sep. 2010 1587


Evaluation of Systemic Follistatin in Pompe Mice
starting at 10, 7, or 5 months of age, i.e., where they demonstrate
different degrees of rotarod dysfunction (Figure 1a). We reasoned
that such a comparison might reveal the impact of pre-existing
myopathy on treatment efficacy. Surprisingly, ­follistatin failed to
improve rotarod performance, either alone or in combination with
GAA, regardless of the age at treatment (Figure 4a–c). Indeed, in
mice treated at 5 months of age, which had up to 90% clearance
of skeletal muscle glycogen and a significant follistatin-mediated
increase in lean mass (Supplementary Figure S2), those treated
with GAA alone tended to perform best (Figure 4c). Thus, despite
measurable follistatin-mediated increases in muscle mass and grip
strength, systemic follistatin failed to rescue their performance on
the rotarod.
Pompe mice treated with follistatin demonstrate
muscle fiber hypertrophy
Follistatin and other myostatin inhibitors reportedly can stimulate
muscle fiber hypertrophy.7,9,10 To determine whether the observed
increase in muscle mass in the follistatin-treated Pompe mice was
similarly due to muscle fiber hypertrophy, cross-sections of quad-
riceps muscle were analyzed for fiber size. The images showed that
the muscle fibers of Pompe mice treated at 7 months of age with
both GAA and follistatin were larger than those treated with GAA
alone or vehicle (Figure 5a,b); these differences were not as appar-
ent in Pompe mice treated with GAA and follistatin starting at
10 months of age (Figure 5c,d).
To quantify the differences in fiber size, 250–500 fibers/mouse
were outlined and the cross-sectional area of each fiber deter-
mined. Results for each treatment group were displayed in terms
of their average fiber area (Figure 6a,b) and fiber size distribution
(Figure 6c,d). When Pompe mice were treated at 7 months of age,
follistatin generated significantly larger muscle fibers compared to
those administered either GAA alone or vehicle (Figure 6a). In the
10-month-old Pompe mice, however, the more modest increase
in fiber size in the group treated with GAA and follistatin only
attained significance when compared to the vehicle-treated mice
(Figure  6b). A proportion of the fibers in Pompe mice treated
with follistatin at 7 months of age were large, with some being
unusually large (>4,000 µm2); vehicle- and GAA alone-treated
animals showed smaller sized fibers and almost none in the very
large size categories (Figure 6c). In contrast, Pompe mice that had
been treated with follistatin at 10 months of age showed little dif-
ference in their fiber size distribution regardless of their treatment
(Figure  6d). These histological data correlate well with the lean
mass and grip strength data noted earlier, i.e., follistatin treatment
stimulated significant muscle fiber hypertrophy in Pompe mice
only when they were treated before 10 months of age.
As the muscle fibers in Pompe mice age, they show an increased
incidence of centralized nuclei.6,28 Central nucleation is also a fea-
ture of other mouse models of muscular dystrophy and reflects
rounds of muscle degeneration and regeneration. In muscular
dystrophy mice, treatment with myostatin inhibitors increased
the percentage of fibers with centralized nuclei, which was attrib-
uted to treatment-stimulated increases in muscle regeneration.7,29
In wild-type mice, very few muscle fibers exhibited a phenotype
characterized by centrally localized nuclei (data not shown). In
contrast, in vehicle-treated Pompe mice at 7 or 10 months of age,
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© The American Society of Gene & Cell Therapy
~35% of fibers had centralized nuclei (Supplementary Figure S3).
Pompe mice treated at either 7 or 10 months of age with follista-
tin had an increased percentage of central nucleated fibers com-
pared to vehicle-treated controls, and this response was somewhat
greater in the mice treated at the younger age. These results suggest
that follistatin may have stimulated some regeneration of Pompe
muscles, even in the older mice.
Discussion
Enzyme replacement therapy was approved by the US Food and
Drug Administration and the European Medicines Agency in
2006 for patients with Pompe disease. Clinical trials in infants
demonstrate improved survival and improvement in hypertro-
phic cardiomyopathy.30–33 A randomized, placebo-controlled,
multicenter trial in adults demonstrated stabilization of musculo-
skeletal outcomes and respiratory parameters.34 The presentation,
natural history, and progression of Pompe is extremely heteroge-
neous, as is the response to treatment, and some patients continue
to show muscle weakness despite enzyme replacement therapy.35,36
Data generated in aged Pompe mice demonstrated incomplete
functional recovery following gene or protein therapy,6,28 which
suggested that the aged Pompe mouse might serve as a model for
patients with poor motor function response. We have used aged
Pompe mice to evaluate the potential of follistatin as an adjuvant
to stimulate muscle cell regeneration and/or hypertrophy. We
hypothesized that generating new muscle tissue with follistatin
in the presence of GAA to treat the defect in glycogenosis would
improve motor function in the aged Pompe mouse.
We constructed an AAV8 vector encoding the secreted iso-
form of human follistatin (FS344),17 which has been shown to bind
to myostatin and inhibit its activity. Intramuscular ­delivery of an
AAV1 vector expressing FS344 effected an increase in the size and
strength of the injected muscles in mdx mice and in primates.11,20
Pilot experiments in wild-type mice following liver-directed
­delivery of our AAV8-follistatin vector demonstrated bioactiv-
ity of the follistatin secreted into the circulation, as illustrated by
increases in total body weight, gross lean mass, and the size of
the quadriceps muscle similar to that reported for intramuscular
delivery.11 In contrast to these effects in wild-type mice, studies in
aged Pompe mice (10 months of age) showed little effect of fol-
listatin on skeletal muscle, despite the somewhat higher circulat-
ing levels (0.4 µg/ml versus 1 µg/ml, respectively). No measurable
increase in gross muscle mass or improved grip strength could
be discerned in mice treated with both AAV8-GAA and AAV8-
follistatin compared to those treated with AAV8-GAA alone.
Histological analysis of the mice in this study did show some evi-
dence of a response to follistatin, viz., an increase in the percent-
age of central nuclei in the follistatin-treated groups and a trend
toward muscle fiber hypertrophy, but these effects did not trans-
late into measurable functional efficacy.
In younger (7-month-old) Pompe mice, however, treatment
with the combination of vectors did increase muscle mass and
strength over GAA treatment alone. We also documented sig-
nificant muscle fiber hypertrophy in response to follistatin in
the 7-month-old mice that was not evident when the mice were
treated at 10 months of age. The positive response to follistatin
in the younger mice was likely due to the fact that there was less
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© The American Society of Gene & Cell Therapy
established myopathy at the time of vector administration. Because
similar levels of transgene expression were documented in both
age groups, a disparity in GAA or follistatin exposure cannot
explain the observed differences in efficacy. Glycogen clearance
following AAV8-GAA treatment of the aged mice was incomplete,
as had been seen previously,6 but substrate levels were not appre-
ciably different between the two age groups being compared.
Previous studies suggested that 10-month-old mice should
be capable of responding to myostatin inhibition. For example,
in a transgenic model, the muscles of 24-month-old Mstn−/−
mice were twice the size, and retained an elevated capacity for
regeneration compared to age-matched normal mice.13 In addi-
tion, administration of a myostatin antagonist increased muscle
regeneration and grip strength in a 24-month-old mouse model
of sarcopenia.14 These results suggest that it is more likely that the
disease-associated muscle pathology in the Pompe mice rather
than age interfered with the response to follistatin.
We also observed that 7-month-old Pompe mice treated with
follistatin alone showed a small increase in lean mass and grip
strength, but the muscles responded more robustly to follistatin
when substrate had been depleted by concomitant GAA adminis-
tration. This result suggests that lysosomal glycogen stores in the
skeletal muscle somehow interfere with a response to follistatin.
These results may not be surprising as the enlarged, glycogen-filled
lysosomes and autophagosomes characteristic of this disease have
been shown to interrupt the contractile apparatus in the muscle of
the 6neo/6neo mouse,3 and interfere in mechanical force generation
in a similar GAA knockout mouse model.37
Despite the significant increases in grip strength measured
in the follistatin-treated Pompe mice described above, we did
not see improvement in rotarod performance, even when treat-
ment was initiated at 5 months of age. In fact, 5-month-old mice
treated with GAA alone tended to perform the best in this func-
tional assay. This observation indicates that muscle function was
not completely normalized by follistatin treatment. It should also
be noted that there was less improvement in grip strength when
hindlimbs were included in the assessment. Although the front
limb grip strength values approached those of normal mice, when
mice were allowed to grip the test apparatus with all of their limbs
only a partial correction of force was achieved. There is typically
more obvious muscle dysfunction in hindlimbs of this mouse
model, as they become splayed and weak as the mice age. Our
data imply that the hindlimb dysfunction was not fully corrected,
which likely affected rotarod performance. Disparity between
muscle hypertrophy and motor function outcomes following myo-
statin inhibition has been reported previously. For example, Qiao
et al.10 demonstrated increased grip strength but reduced tread-
mill endurance in mdx mice treated with ­myostatin ­propeptide.
Amthor et al.38 described an increase in muscle mass that did not
translate to a corresponding increase in specific force in myosta-
tin knockout mice, and postulated that there are qualitative dif-
ferences in the muscle generated during myostatin deficiency,
e.g., changes in fiber type distribution and a reduced number of
mitochondria.
Others have demonstrated a dramatic expansion of autopha-
gosomes leading to centralized cores of cellular debris in type II
muscles of Pompe mice.4 Similar findings have been described
Molecular Therapy vol. 18 no. 9 sep. 2010
Evaluation of Systemic Follistatin in Pompe Mice
in muscle fibers from Pompe patients.5 The associated disorga-
nized microtubular network and reduced cycling of the cation-
­independent mannose-6 phosphate receptor (required for GAA
uptake) interfered with trafficking of GAA to the lysosomes,
and led to the subsequent resistance of these damaged fibers to
­glycogen clearance.4,39 Based on our observations, we contend
that as this muscle pathology progresses in the Pompe mouse, the
affected fibers become unresponsive to follistatin due to the reten-
tion of glycogen and the accumulation of autophagic debris. We
had previously observed thickening of the endomysium and mild
fibrosis in the muscles of older Pompe mice.6 Although we did
not specifically evaluate levels of fibrosis in the current studies,
this pathology could also have contributed to a lack of functional
response to follistatin in the older mice. Thus, these other patho-
logic features, in addition to glycogen storage per se, could con-
tribute to the incomplete functional correction observed in our
studies, particularly in the 10-month-old Pompe mice.
In summary, we did not achieve our original goal of improv-
ing function in 10-month-old Pompe mice by using systemic
­follistatin as an adjuvant to GAA treatment. We were able to show
some positive effects of follistatin in younger Pompe mice, and
this response was more significant when glycogen stores in the
skeletal muscles were reduced. However, even in the younger mice
motor dysfunction was not completely alleviated. It is difficult to
predict whether patients might respond positively to treatment
with an adjuvant like follistatin, as histopathology of muscle from
Pompe patients before and after enzyme replacement therapy
showed a variable response in terms of glycogen clearance.40,41
Importantly, our data imply that patients will be more likely to
benefit from either GAA or muscle-enhancing molecules like
­follistatin if treated early, before irreversible ­muscle pathology
has developed.
Materials and Methods
Construction of plasmids and vectors. A synthetic human follistatin-344
complementary DNA (FST344 GenBank accession NM_013409) was gen-
erated with 5′ EcoRI and 3′ SfiI sites and subcloned into pUC (Genescript,
Piscataway, NJ). Plasmid pDC190-follistatin was generated by ligating
the 1,140-bp follistatin complementary DNA into the EcoRI/SfiI sites of
pDC190.25 This put follistatin expression under the control of a hepato-
cyte-restricted human serum albumin promoter (DC190). The follistatin
expression cassette was then subcloned into the MfeI–KpnI sites of the
AAV2 previral vector pAAV/SP70. Due to viral packaging issues, pAAV/
SP70-follistatin was modified. Plasmid pAAV/SP70-follistatin was cut
with KpnI, blunt ended with Klenow and cut with SfiI. A 1,367-bp frag-
ment of the human α1-antitrypsin 3′ intron stuffer sequence was generated
from PmlI and SfiI digest of pAAV/SP70-IGF142 and ligated into pAAV/
SP70-follistatin. The DC190-follistatin vector DNA was packaged into
AAV8 capsid using standard triple plasmid transfection into HEK 293
cells. The AAV8/DC190-follistatin (AAV8-follistatin) virus was purified
by iodixanol-gradient centrifugation followed by ion exchange chromato­
graphy over Hitrap Q HP column. AAV8/DC190-GAA (AAV8-GAA) has
been previously described.6 Virus titers were determined by real time PCR
with primers to the bovine growth hormone polyadenylation sequence,
and quantified as DNase-resistant particles.25
Animal studies. The Pompe6neo/6neo (Pompe) mice used in these studies have
a disruption of exon 6 in the GAA gene resulting in a lack of enzyme activi-
ty.3 Pompe, 129S2/SvPasCrl (129S2), C57BL/6NCrl (C57B6) (Charles River
Laboratories, Waltham, MA), and B6.129SF2/J (B6.129) (Jackson Laboratory,
1589

Evaluation of Systemic Follistatin in Pompe Mice
Bar Harbor, ME) were housed in small groups with free access to food and
water in an AAALAC accredited facility. All studies were reviewed and
approved by the institutional animal care and use committee. Test articles
were administrated via tail-vein injection in 0.2–0.3 ml. Blood samples were
collected from the orbital venous plexus under anesthesia (3% isoflurane)
using microhematocrit capillary tubes (VWR, Bridgeport, NJ), and collected
into BD gold microtainer tubes (VWR). Serum was separated by centrifuga-
tion at 10,000g for 10 minutes and stored at −80 °C. Once a month animals
were transferred from the housing area into separate rooms for functional
testing or measurement of body mass index (see below). At the end of each
study, mice were killed by CO2 asphyxiation or intraperitoneal injection of
Euthasol (pentobarbital sodium and phenytoin sodium; Virbac, Fort Worth,
TX), after which tissues were removed and either fixed in 10% neutral-
­buffered formalin or snap frozen on dry ice and stored at −80 °C for later use.
In some cases, individual dissected tissues were weighed before processing.
It should be noted that a large percentage of the mice treated with
AAV8-follistatin developed prolapsed rectums between 3 and 6 months
postadministration. This was seen in Pompe mice treated with follistatin
alone, or a combination of follistatin and GAA, but was not observed in
any GAA or vehicle-treated mice. Prolapsed rectums were also observed
in wild-type mice treated with follistatin, indicating that this was not a
consequence of the Pompe disease phenotype. This effect seemed to
be somewhat dose-dependent as we did not observe prolapses in mice
that had lower follistatin expression levels (0.1 compared to 0.5–2 µg/ml
serum). It is possible that this side effect is follistatin specific, and not a
general response to myostatin inhibition, as follistatin has been shown
to bind several members of the transforming growth factor-β family of
regulatory proteins in addition to myostatin,17–19 which can inhibit activin
signaling in a variety of tissues (for a review see refs. 43,44).
Functional assays and measurement of body mass index. The 6neo/6neo
Pompe mice have both C57B6 and 129S2 genetic background. Both of these
strains, and a B6.129 cross, were used as wild-type control strains in the
evaluation of functional assays. Muscle coordination and endurance were
assessed on a rotarod using the Smartrod program (AccuScan Instruments,
Columbus, OH). Mice were placed on a 30-mm diameter spindle that
was elevated 18 inches. The spindle was programmed to accelerate from
0–30 r.p.m. over 90 seconds. The time to fall (latency) was recorded auto-
matically with a light beam sensor at the bottom of the apparatus. Each
animal was subjected to three trials with ~5-minutes rest between trials
and the average latency was determined. Muscle strength was tested using
a Chatillon grip strength meter (Columbus Instruments, Columbus, OH).
Mice were encouraged to grip a wire grid attached to the force meter with
their front limbs alone, or with both the front and rear limbs. Once a firm
grasp had been established the animals were pulled away until they lost
their grip. The maximal force generated was recorded electronically, and
the average from two trials per time point was reported. It should be noted
that these functional assays show some study-to-study variability and
are best evaluated against internal control groups, i.e., ­quantitative com-
parisons between studies are not always valid. During the course of the
studies animals were weighed (weekly or monthly) and their whole‑body
mass composition measured using an EchoMRI 3-in-1 Whole Body
Composition Analyzer (Echo Medical Systems, Houston, TX), which uses
nuclear magnetic resonance to determine lean and fat mass.
Measurement of glycogen, GAA, and follistatin levels. Frozen tissue
samples were weighed and suspended in sterile water at a concentration
of 100 mg/ml in a 2-ml microcentrifuge tube. A 5-mm stainless steel bead
(Qiagen, Valencia, CA) was added to the tube and the tissue was disrupted
using a TissueLyser (Qiagen) at 30 Hz for 10 minutes in a cold room (4 °C).
Samples were stored frozen at −80 °C, thawed, centrifuged at 14,000 g for
15  minutes at 4 °C, and aliquots stored at −80 °C. To measure glycogen,
lysates were defrosted and digested with α-amylglucosidase (0.54 mg/ml;
Sigma-Aldrich, St Louis, MO) in 25 mmol/l potassium acetate buffer, pH
1590
© The American Society of Gene & Cell Therapy
5.5. Glucose released from tissues, and the levels of endogenous glucose
from parallel undigested samples, were quantitated with the Amplex Red
glucose/glucose oxidase assay kit (Molecular Probes, Eugene, OR) accord-
ing to manufacturer’s instructions. Glycogen levels were determined by
subtracting the undigested glucose levels from the digested samples. Bovine
liver glycogen (Sigma-Aldrich) was used as a reference standard. Serum
GAA levels were determined using an enzyme-linked immunosorbent
assay as previously described.6 Serum follistatin levels were determined
using an hFollistatin enzyme-linked immunosorbent assay (R&D Systems,
Minneapolis, MN).
Histopathology. Mouse quadriceps were fixed in 10% neutral-buffered
formalin (VWR) and embedded in paraffin. Five-micron sections were
cut and stained with hematoxylin–eosin. All slides were scanned at an
absolute magnification of ×200 using the Aperio ScanScope XTsystems
(Aperio Technologies, Vista, CA). The background illumination ­levels
were ­calibrated using a prescan procedure. All acquired images were
subsequently labeled, placed in dedicated project folders, and stored on
a local server. Slides were viewed and analyzed remotely using desktop
personal computers employing the web-based ImageScope viewer (Aperio
Technologies). To evaluate the mean and distribution of muscle fiber sizes,
outlines were manually drawn around the periphery of individual fibers
and the cross-sectional area calculated automatically by the software, then
the data were exported into Excel (Microsoft, Redmond, WA) for further
analysis. Between 250 and 500 fibers/mouse were measured. To evaluate
fiber size distribution, fibers were grouped into nine size categories and the
percentage of fibers that fell within each category was determined for the
different treatment groups.
Statistical analysis. All data are reported as means with corresponding
SEM. Studies comparing multiple groups were evaluated using analysis of
variance, followed by Bonferroni’s multiple comparison post-test. When
only two groups were compared, Student’s t-test was used for analysis.
Statistics were performed using GraphPad Prism software (GraphPad
Software, La Jolla, CA). A P value of <0.05 was considered statistically
significant.
SUPPLEMENTARY MATERIAL
Figure S1.  Pompe mice treated with follistatin at 7 months of age
showed an increase in quadriceps mass.
Figure S2.  Glycogen levels and lean mass measurements from Pompe
mice treated with follistatin at 5 months.
Figure S3.  Histological evaluation of muscle fiber regeneration.
Materials and Methods.
ACKNOWLEDGMENTS
We thank members of Comparative Medicine, Virus Production, and
Histology; as well as Nelson Yew, Jennifer Nietupski, Alida D’Angona, Bill
Weber, and Nilesh Pande for technical assistance. We also ­acknowledge
Brad Hodges, Allison McVie-Wylie, Rod Moreland, Jonathan Fidler, Lisa
Stanek, and Brian Kasper (Ohio University) for help with experimen-
tal design. For critical review of the manuscript, we thank Ed Kaye,
Joan Keutzer, and Jennifer Tousignant. All of the authors are employees
of and shareholders in Genzyme Corporation.
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