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A Phase I/IIa Follistatin Gene Therapy Trial for Becker Muscular Dystrophy.


Abstract and Figures

Becker muscular dystrophy (BMD) is a variant of dystrophin deficiency resulting from DMD gene mutations. Phenotype is variable with loss of ambulation in late teenage or late mid-life years. There is currently no treatment for this condition. In this BMD proof-of-principle clinical trial, a potent myostatin antagonist, follistatin (FS), was used to inhibit the myostatin pathway. Extensive pre-clinical studies, using adeno-associated virus (AAV) to deliver follistatin, demonstrated an increase in strength. For this trial, we used the alternatively spliced FS344 to avoid potential binding to off target sites. AAV1.CMV.FS344 was delivered to 6 BMD patients by direct bilateral intramuscular quadriceps injections. Cohort 1 included 3 subjects receiving 3X10(11) vg/kg/leg. The distance walked on the 6MWT was the primary outcome measure. Patients 01 and 02 improved 58 meters (m) and 125 meters, respectively. Patient 03 showed no change. In Cohort 2, Patients 05 and 06 received 6X10(11) vg/kg/leg with improved 6MWT by 108 m and 29 m, whereas, Patient 04 showed no improvement. No adverse effects were encountered. Histological changes corroborated benefit showing reduced endomysial fibrosis, reduced central nucleation, more normal fiber size distribution with muscle hypertrophy, especially at high dose. The results are encouraging for treatment of dystrophin-deficient muscle diseases.Molecular Therapy (2014); doi:10.1038/mt.2014.200.
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original article
© The American Society of Gene & Cell Therapy
Becker muscular dystrophy (BMD) is a variant of dys-
trophin deficiency resulting from DMD gene mutations.
Phenotype is variable with loss of ambulation in late
teenage or late mid-life years. There is currently no treat-
ment for this condition. In this BMD proof-of-principle
clinical trial, a potent myostatin antagonist, follistatin
(FS), was used to inhibit the myostatin pathway. Exten-
sive preclinical studies, using adeno-associated virus
(AAV) to deliver follistatin, demonstrated an increase in
strength. For this trial, we used the alternatively spliced
FS344 to avoid potential binding to off target sites. AAV1.
CMV.FS344 was delivered to six BMD patients by direct
bilateral intramuscular quadriceps injections. Cohort 1
included three subjects receiving 3×1011 vg/kg/leg. The
distance walked on the 6MWT was the primary outcome
measure. Patients 01 and 02 improved 58 meters (m)
and 125 m, respectively. Patient 03 showed no change.
In Cohort 2, Patients 05 and 06 received 6×1011 vg/kg/
leg with improved 6MWT by 108 m and 29 m, whereas,
Patient 04 showed no improvement. No adverse effects
were encountered. Histological changes corroborated
benefit showing reduced endomysial fibrosis, reduced
central nucleation, more normal fiber size distribution
with muscle hypertrophy, especially at high dose. The
results are encouraging for treatment of dystrophin-
deficient muscle diseases.
Received 26 August 2014; accepted 8 October 2014; advance online
publication 18 November 2014. doi:10.1038/mt.2014.200
Becker muscular dystrophy (BMD) is a clinical variant of dystro-
phin deciency of muscle caused by a DMD gene mutation. e
clinical course of BMD is milder compared to Duchenne muscu-
lar dystrophy (DMD), but there is wide variability in phenotype.
ere may be a delay in motor development, however in most
cases reported symptoms relate to participation in sports in early
teenage years. Lost ambulation is a major milestone that occurs
in the fourth or h decade, although wheelchair independence
may be preserved until aer age 60.1 Cardiomyopathy is oen the
cause of death in BMD related to severe le ventricular dilation
with reduced ejection fraction, complicated by life-threatening
arrhythmias.2 e majority of BMD patients have deletions of the
DMD gene, estimated at a frequency of 80%.3 Other BMD caus-
ing mutations include missense mutations,4 exon duplications,5
and even out-of-frame exon deletions or nonsense mutations that
predict no signicant dystrophin translation.6,7 Attempts to dene
the clinical course by dystrophin on muscle biopsy have been
For clinical trials, there is consensus that distinction of BMD
from DMD relies not on the specic mutation or dystrophin pro-
tein levels on muscle biopsy, but rather on the ability to maintain
ambulation beyond age 16 years.7,10,11 Another key feature of the
ambulatory BMD patient is the targeted weakness of the quadri-
ceps muscles (knee extensors).10,12,13 is can be relatively selec-
tive, so much so that it manifests as a form fruste, referred to as
quadriceps myopathy.14 Oen it is this selective lower extremity
weakness that predisposes patients to frequent falls and is a key
determinate in maintaining independent ambulation. Increasing
muscle strength in BMD is challenging and no treatment modality
has been identied.15,16 Of interest, the benet of glucocorticoids
as demonstrated for the dystrophinopathy in the DMD population
has not proved eective in BMD.17 In the current clinical trial, a
potential strategy to achieve a clinically meaningful eect on mus-
cle health and strength was applied to BMD through inhibition of
the myostatin pathway. Extensive studies in the mdx mouse18 and
in nonhuman primates19 supported this approach, demonstrating
signicant increases in strength by delivery of follistatin (FS) using
adeno-associated virus (AAV). FS has been shown to function as a
potent myostatin antagonist with the additional benet of control-
ling muscle mass through pathways independent of the myostatin
signaling cascade.20 ere are two isoforms of follistatin generated
by alternative splicing and initially translated to isoforms FS317
and FS344.21 Posttranslational modication of each cleaves a 29
amino acid signal peptide giving rise to FS288 and FS315. FS288
functions collaboratively in reproductive physiology with activin
Follistatin Gene erapy Trial in BMD
Molecular erapy
original article
© e American Society of Gene & Cell erapy
Correspondence: Jerry R Mendell, Gene Therapy Center Nationwide Children’s Hospital, Columbus, OH 43205, USA.
A Phase 1/2a Follistatin Gene Therapy Trial for
Becker Muscular Dystrophy
Jerry R Mendell1,2,3, Zarife Sahenk1,2,3, Vinod Malik1, Ana M Gomez1, Kevin M Flanigan1,2,3,
Linda P Lowes2,4, Lindsay N Alfano2,4, Katherine Berry2,4, Eric Meadows1, Sarah Lewis1, Lyndsey Braun1,
Kim Shontz1, Maria Rouhana1, Kelly Reed Clark1,2, Xiomara Q Rosales1,2, Samiah Al-Zaidy1,2,
Alessandra Govoni1, Louise R Rodino-Klapac1,2, Mark J Hogan5 and Brian K Kaspar1,2
1Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, Ohio, USA; 2Department of Pediatrics, The Ohio State University, Columbus,
Ohio, USA; 3Department of Neurology, The Ohio State University, Columbus, Ohio, USA; 4Department of Physical Medicine and Rehabilitation, The
Ohio State University, Columbus, Ohio, USA; 5Department of Radiology, Vascular and Interventional Radiology, Nationwide Children’s Hospital,
Columbus, Ohio, USA.
Molecular Therapy 1
© The American Society of Gene & Cell Therapy
Follistatin Gene Therapy Trial in BMD
and inhibins of the hypothalamic pituitary-gonadal axis.22 FS315
more reliably targets skeletal muscle, has no known cardiotoxicity
or other adverse eects and is ideal for gene delivery to muscle.
AAV1.CMV.FS344 delivered by direct intramuscular injec-
tion to quadriceps and tibialis anterior muscles of the mdx mouse
increased muscle mass and strength throughout the lower extrem-
ities with a demonstrable remote eect on these same parameters
in the upper limbs and increased muscle mass in the paraspinal
muscles.18 is we attributed to the muscle acting as a secretory
site for follistatin with the circulating isoform reaching remote
sites.23 AAV1.CMV.FS344 was further tested in the nonhuman
primate to explore a paradigm applicable to clinical trial. In the
cynomolgus macaque, we injected AAV1.FS344 directly into the
quadriceps muscle resulting in an increase in size and strength
of this muscle.19 ese preclinical studies in the absence of toxic-
ity enabled a phase 1/2a clinical trial in patients with BMD (IND
Patient characteristics and response to treatment
Six male BMD patients were treated according to a dose-ascend-
ing gene therapy regimen (Table 1 ). is was a single site study
conducted at Nationwide Children’s Hospital. Enrolled subjects
were ambulatory with knee extensor muscle weakness greater
than 2 SDs below age expectations.24 Participants were not on any
immunosuppressive therapy at the time of recruitment, but were
placed on prednisone 1 month prior to AAV1.CMV.FS344 injec-
tions as a precaution against an immune response to AAV cap-
sid, as previously found in human clinical trials.25–27 Prednisone
dosing remained the same for ~1 month postinjection and was
tapered o by day 60 postgene delivery. T cell responses towards
AAV1 capsid and follistatin were assessed by IFN-γ ELISpot assay
and were <50 spot forming cells/million PBMCs for each par-
ticipant upon enrollment. Serum neutralizing antibody titers to
AAV1 were assessed by ELISA and were below 1:50 at the start
of the study and monitored according to a previously published
clinical trial schedule.26,27 Muscle biopsies were performed 30 days
prior to administration of AAV1.CMV.FS344 as a baseline histo-
pathological assessment of muscle with a follow up biopsy on the
opposite extremity at day 180 postgene transfer. e extremity
undergoing initial biopsy was chosen by a randomization table
and taken from the proximal vastus lateralis, thus determining the
postbiopsy site in the opposite extremity targeting the same head
of the quadriceps. Serum chemistry/hematology batteries were
assessed at baseline, days 7, 14, 30, 60, 90, 180, and 1 year to evalu-
ate for adverse eects due to gene transfer and included: complete
blood count, liver function studies, kidney function (cystatin C),28
amylase, creatine kinase, and serum hormones (FSH, LH, testos-
terone, estrogen).
Cohort 1 included three ambulatory subjects, ages 30, 35,
and 37 (34±3.6), genetically diagnosed with in-frame DMD gene
mutations. Subjects in this cohort received 3×1011 vg/kg/leg (total
6e11 vg/kg/patient) delivered to three of the four muscle groups of
the quadriceps: the vastus lateralis (VL), rectus femoris (RF), and
vastus medialis (VM). Four injections were delivered per muscle,
each with the guidance of ultrasonography and a MyoJect Luer
Lock EMG needle. is rst cohort has now been followed for 1
year postgene delivery (Figure 1). In two subjects, improvement
on the 6MWT was robust: Patient 01 improved by 58 meters (m),
and Patient 02 by 125 m. Patient 03 improved modestly, with an
increase of 9 m; however, we would not consider this outside the
range of variability for the BMD population, based on previous
clinical experience. Although, no comparative natural history data
of the 6MWT in BMD patients is available, substantial increases
in 6MWT as observed in our subjects would not be predicted over
the course of 1 year in untreated BMD patients.
Furthermore, the improvement in walk distance in patient’s
01 and 02 cannot be attributed to prednisone, since they had
completely stopped the drug by day 90, while strength peaked
at day 180 and was maintained throughout the remainder of
the clinical trial. ere were no signicant adverse events dur-
ing this trial that were related to gene transfer (Supplementary
Table S1). No abnormalities were noted in any organ system
assessment of liver, kidney, or bone marrow, and pituitary-
gonadal hormone levels (FSH, LH, estrogen, testosterone
(Supplementary Figure S1)) remained normal throughout the
trial. Assessment of the IFN-γ ELISpot assay for T-cell immune
responses to AAV1 capsid or follistatin showed no consistent
or predictable response related to T-cell immunity between
patients (Figure 2). Of particular note, Patient 03 who achieved
the least benet in this cohort from gene transfer showed vir-
tually no increase in T-cell immunity throughout year 1, while
Patient 02 showed an increase in T cells targeting AAV1, and
patient 01 showed increased T cells to follistatin. Serum anti-fol-
listatin antibody levels were never elevated above pretreatment
levels (remained below 1:50 titer) in Cohort 1.
Based on the safety of Cohort 1, an additional three BMD
patients were enrolled in the ascending dose trial. Cohort 2
included ambulatory subjects ages 24, 30, and 34 (29±5.0) with
in-frame DMD gene mutations (Tabl e 1 ). e dose for this group
was increased to 6×1011 vg/kg/leg (1.2e12 vg/kg/patient). Gene
delivery followed the paradigm described for the rst cohort with
delivery to the three major groups of the quadriceps: VL, RF, and
VM. ese three patients (04, 05, and 06) have now been followed
for 6 months and the results of the 6MWT are shown in Figure1.
It is likely that Cohort 2 subjects have received maximum benet
from gene transfer based on ndings in the rst cohort. Subject
one of Cohort 2 (Patient 04) showed the least benet of any
patient in the trial. ere was a decrease in the 6MWT by 14 m.
Table 1 Characteristics of becker muscular dystrophy patients enrolled
in trial
Cohort 1 01 30 del exon 48–49
(3×1011 vg/kg per leg)
02 35 point mutation exon 8a
03 37 del exon 45–48
Cohort 2 04 34 del exon 45–48
(6×1011 vg/kg per leg)
05 24 del exon 45–47
06 30 del exon 13
AAV1, adeno-associated virus serotype 1; CMV, cytomegalovirus; del, deletion;
FS344, follistatin isoform 344; vg, vector genome.
aSubexonic deletion (c.676_678delAAG, p.226delLys) in exon 8 of the DMD
© The American Society of Gene & Cell Therapy Follistatin Gene Therapy Trial in BMD
e other two patients in this cohort improved their walking dis-
tance. Patient 05 increased by 108 m, and Patient 06 by 29 m, with
improvements found as early as 1 month postgene delivery and
maintained over 6 months.
In neither cohort did we nd a consistent increase in quad-
riceps muscle strength following AAV.FS344 gene transfer. is
nding follows a pattern we encountered in our clinical trial of
eteplirsen for exon skipping where we also showed functional
benet in the 6MWT without increasing quadriceps strength over
a similar duration of study.29 We believe that muscle brosis is a
barrier to increasing quantitative measures of muscle strength in
single muscle groups, accounting for the poor correlation. e
success we report here is related to follistatin gene therapy target-
ing a composite group of muscles contributing to the results of
6MWT because of the remote eect of secretion following FS344
transduced muscle bers. Remote eects of AAV1.CMV.FS344
were apparent in preclinical studies in both mice and nonhuman
primates.18,19 Another factor contributing to outcomes was pre-
dicted by McDonald et al suggesting that longer duration stud-
ies may be necessary to increase absolute values of strength by
myometr y. 30
In Cohort 2 subjects as in the low dose subjects, no signi-
cant adverse events were encountered (Supplementary TableS1),
serum chemistries and hormone levels (Supplementary
Figure S1) remained normal, and there was there was no con-
sistent pattern of T-cell immunity specic to AAV capsid pool as
evaluated by ELISpot assays (Figure 2). Patient 06 showed early
and signicant elevation of immune response to follistatin that
was not present prior to gene transfer. Serum anti-follistatin anti-
body levels in Cohort 2 remained below 1:50 titers.
Gross examination and MRI results
Our goal at the conceptualization of this clinical trial was to dif-
fusely and symmetrically increase the size of the quadriceps
muscle. Muscle hypertrophy was an outcome we had seen in
mice and nonhuman primate studies injected with AAV1.CMV.
FS344, in a manner that extended well beyond the specic sites
of injection.18,19 In the cynomolgus macaque, each of the three
major muscles of the quadriceps (VL, RF, and VM) received a
single injection. Follistatin secretion from transduced muscle
at the site of injection reached remote sites. In the clinical trial,
we compensated for the larger muscle mass by distributing four
injections to each of three major muscle groups of the quadriceps.
However, despite ultrasound-guided injections designed to target
muscle and avoid regions of muscle brosis, this was only possible
up to a degree. Two subjects with strikingly dierent degrees of
muscle brosis illustrate the challenge (Figure 3ad) of eectively
delivering AAV1.CMV.FS344 to muscle. For example, Patient 06
(Figure 3a,b) showed signicant improvement in 6MWT (108 m)
and had less muscle brosis compared to Patient 03 (Figure 3c,d)
who exhibited no signicant improvement in the 6MWT (9 m).
Subsequent analysis using an MRI-based grading scale applied
to thigh muscles at the time of enrollment conrmed brosis as
a major obstacle in achieving improved 6MWT (Figure 4). It is
apparent that muscle brosis precluded the diuse follistatin-
induced muscle hypertrophy that we had seen in the normal
muscle of the nonhuman primate. Of note, in this clinical trial,
gross muscle hypertrophy was focal following gene transfer and
could be observed on clinical examination (Figure 5). ese areas
of muscle were strikingly apparent and oen pointed out by the
Figure 1 Distance walked in 6-minute walk test (6MWT) following follistatin gene therapy. (a) Distance walked in meters in the 6MWT for
subjects receiving AAV1.CMV.FS344 in each leg (3×1011 vg/kg/leg) with follow up for 1 year. A stippled red line shows the baseline for each patient.
Patients are numbered consecutively based on treatment at ~4–6 week intervals. (b) The table shows the exact distances at each time point from
baseline (BL) to 1 year. The “12-mo change” indicates the distance walked compared to BL. (c) Distance walked in meters in the 6MWT for subjects
receiving AAV1.CMV.FS344 in each leg (6×1011 vg/kg/leg) with follow up for 6 months. (d) The table again shows the exact distances at each time
point from baseline (BL) to 6 months. The “6-mo change” indicates the distance walked compared to BL. D, day.
6MWT (meters)
6MWT (meters)
Low dose cohort
01 02 03
BL 492 291 457
D30 491 314 464
D60 511 329 468
D90 525 386 455
D180 550 401 470
1 YR 550 416 466
12-mo change +58 m +125 m +9 m
High dose cohort
04 05 06
BL 439 515 452
D30 437 574 477
D60 427 570 469
D90 434 600 475
D180 425 623 481
6-mo change -14 m +108 m +29 m
30 days
60 days
90 days
6 months
30 days
60 days
90 days
6 months
1 year
Molecular erapy 3
© The American Society of Gene & Cell Therapy
Follistatin Gene Therapy Trial in BMD
Muscle biopsy analysis
To further evaluate the eects of AAV1.CMV.FS344, we per-
formed muscle biopsy analyses comparing pre- and posttreat-
ment muscle biopsies obtained 30 days prior to gene delivery
and at 6 months following gene transfer. One patient refused a
second biopsy (Patient 04) and another showed severe brosis
in the area targeted for the second biopsy (Patient 03) limiting
interpretation. Postinjection biopsies from the low dose subjects
(Cohort 1; Patient 01 and 02) highlighted follistatin-induced
regeneration.31–33 e biopsies demonstrated an increase in
the number of muscle bers per unit area, inclusive of small
and medium size diameter subpopulations (Supplementary
FigureS2a). e ndings favor improved radial growth of small
bers resulting from enhanced muscle regeneration combined
with decreased frequency of necrosis/regeneration cycles in the
muscle. e follistatin eect was better dened in the postin-
jection muscle biopsies from the high dose subjects (Cohort 2,
Patient 05 and 06) (Figure 6ad; Supplementary Figure S2b,c).
ere was a shi to a larger mean ber diameter population:
Patient 05, prebiopsy 40.14±2.10 µm (n = 323 bers) versus
postbiopsy 59.33±1.54 µm (n = 292 bers); P < 0.0001; Patient
06, pre 47.48±2.00 µm (n = 245 bers) versus post 63.74±2.45
µm (n = 277) P < 0.0001. Posttreatment muscle bers appeared
to be more uniform in size distribution distinct from untreated
Becker muscle where many small and hypertrophied bers are
seen side-by-side (Figure 6a,c). More notable, the quantica-
tion of endomysial connective tissue (brosis) using picrosirius
staining conrmed the anti-brotic eect of follistatin previ-
ously reported muscle,33 lung,34 liver,35 and pancreas.36 We found
that the connective tissue was signicantly decreased in post-
treatment biopsy samples from all patients (P < 0.0002, one-
way analysis of variance followed by Bartlett’s test). In Cohort
2 patients posttreatment, we found that connective tissue was
reduced to 35% of baseline for Patient 05 and to 43% of base-
line for patient 06 (P < 0.017, one-way analysis of variance)
(Figure 7, Supplementary Figure S3). In addition, following
Figure 2 Interferon-gamma (IFN-γ) ELISpot assays. The T cell immune responses to AAV1 capsid and follistatin are shown for each patient through-
out the clinical trial. Spot forming cells (SFCs) per million peripheral blood mononuclear cells (PBMCs) are shown on the Y-axis, and days postinfection
(dpi) on X-axis.
01 04
02 05
03 06
SFCs/1e6 PBMCs
BL 714306075
90 180
SFCs/1e6 PBMCs
SFCs/1e6 PBMCs
BL 714306045 75
90 180
SFCs/1e6 PBMCs
SFCs/1e6 PBMCs
BL 714306045
90 180
BL 714306045 75
90 180
Follistatin Pool 1
Follistatin Pool 2
AAV1 Capsid Pool 1
AAV1 Capsid Pool 2
AAV1 Capsid Pool 3
194270 360
BL 714306077
90 180194 270360
BL 714306077
90 180194 270360
© The American Society of Gene & Cell Therapy Follistatin Gene Therapy Trial in BMD
gene transfer both cohorts showed a decrease in the percent of
bers with central nuclei (Supplementary Figure S4) suggesting
that myonuclei movements toward periphery were completed.
DNA copy number at the site of biopsy is shown for each patient
undergoing posttreatment in Supplementary Table S2. Muscle
transgene expression specic for the FS344 isoform by RT-PCR
was corroborated comparing pre- and posttreatment muscle
biopsies (Supplementary Figure S5).
A potential interesting nding in this study was the number
of Pax7+ satellite cell nuclei between pre- and postgene therapy
biopsies. ere has been ongoing concern raised by several inves-
tigators regarding myostatin inhibition and relation to satellite
cell depletion.37–39 In this study comparing pre- and postfollistatin
biopsies there was no consistent decline in the number of Pax7+
satellite cell nuclei per muscle ber (Supplementary Figure S6)
and the quantication of Pax7+ satellite cell nuclei postgene trans-
fer consistently exceeded our previously reported control num-
bers (0.065±0.006).40
Expression of microRNAs in response to follistatin
Previous studies have shown that AAV encoding follistatin
reduces expression of miR-206, miR-1, and miR-29a.41 As a con-
rmatory biomarker for a follistatin eect, we compared miR-206
expression levels between rst and second muscle biopsies from
both cohorts following AAV1.CMV.FS344 injection. In BMD
muscle, in which perpetual necrosis/regeneration cycles take
place, the baseline miR-206 levels were found 4- to 5.6-fold higher
than control muscle samples (Supplementary Figure S7a). Six
months postgene injections there was a down regulation of miR-
206 expression in all patients suggesting an overall slower rate of
necrosis/regeneration events. Similar trends of down regulation
of miR-1 and miR-29c were observed in posttreatment samples
(Supplementary Figure S7b,c).
A solid rationale preceded our clinical trial of follistatin gene deliv-
ery for BMD. A compelling justication is the lack of treatment
Figure 3 Site of gene transfer on leg compared to areas of fibrosis.
(a) The sites of gene transfer to the right leg is shown for Patient 05
(distance walked = 108 m, 6MWT) using a surgical marking pen; (b)
MRI of quadriceps muscles for Patient 05 shows a mild degree of MRI
intensity (T1-weighted image); (c) the sites of gene transfer to the right
leg is shown for Patient 03 (distance walked = 9 m, 6MWT) using a sur-
gical marking pen; (d) MRI of quadriceps muscles for Patient 03 shows
marked increase in intensity indicative of fibrosis.
Figure 4 Grading Scale for quadriceps muscles by magnetic reso-
nance images (MRI). Muscle MRIs were used to establish a grading
scale for the quadriceps muscles based on approximate percentage of
increased image intensity indicating degree of fibrosis replacing normal
muscle. There was an overall correlation between fibrosis and distance
walked on the 6MWT with Patients 03 and 04 demonstrating the least
benefit from gene transfer. RF, rectus femoris; VL/VI, vastus lateralis/vas-
tus intermedius; VM, vastus medialis.
MRI muscle score
01 02 03 04
05 06
Figure 5 Focal areas of clinical muscle hypertrophy. Following gene
transfer, focal areas of muscle hypertrophy (red arrows) could be seen
clinically, as shown in Patients 01 and 05. We never observed diffuse
quadriceps muscle enlargement as we had seen in preclinical studies in
the nonhuman primate.
Molecular erapy 5
© The American Society of Gene & Cell Therapy
Follistatin Gene Therapy Trial in BMD
for this form of muscular dystrophy including failed trials of
glucocorticoids,17 creatine monophosphate,42 sildenal15 and an
attempt to replace dystrophin using a plasmid-based gene replace-
ment strategy.43 e motivation for employing an inhibitor of the
myostatin pathway originated from both preclinical and clinical
studies. e potential importance of this pathway was rst illus-
trated in 1997 in the myostatin knock out mouse showing a large
and widespread increase in skeletal muscle mass.44 Myostatin, a
member of the transforming growth factor-β superfamily, is an
endogenous inhibitor of muscle growth. e eect of myostatin is
conserved throughout mammalian species,45–48 including humans
where the identication of myostatin gene mutations led to hyper-
muscularity through the combination of muscle ber hyperplasia
and hypertrophy.49,50 e benets of loss of myostatin activity are
also well established in dystrophic mice.51–53 e results of the rst
clinical trial of myostatin inhibition using a recombinant neutral-
izing antibody to inhibit myostatin (MYO-029) are likewise of
interest showing a small, dose-related increase in muscle mass
preferentially targeting BMD subjects in preference to other forms
of dystrophy including limb girdle and facioscapulohumeral mus-
cular dystrophies. However, no direct clinical benet in muscle
strength or function was seen in the MYO-029 trial.54
Follistatin is a potent inhibitor of the myostatin pathway and
transgenic mice overexpressing follistatin demonstrate striking
increases in muscle mass.55 e potential for follistatin as a thera-
peutic vehicle is enhanced because of its independence from the
myostatin pathway. In the myostatin-null mouse, follistatin trans-
gene expression results in an impressive quadrupling of muscle
mass.20 In moving to a clinical trial, dening the follistatin isoform
Figure 6 Muscle biopsy changes following follistatin gene therapy. (a) Pretreatment biopsy from Patient 05; (b) Posttreatment biopsy from
Patient 05; (c) Pretreatment biopsy from Patient 06; (d) Posttreatment biopsy from Patient 06. The posttreatment biopsies show reduced fibrosis and
a decrease in central nucleation. The number of small muscle fibers is markedly reduced and fewer split fibers are seen. Fiber size analyses showed a
shift toward larger mean fiber diameter populations: Patient 05, prebiopsy 40.14±2.10 µm (n = 323 fibers) versus postbiopsy 59.33±1.54 µm (n =
292 fibers); P < 0.0001; Patient 06, pre 47.48±2.00 µm (n = 245 fibers) versus post 63.74±2.45 µm (n = 277) P < 0.0001.
30 µm 30 µm
30 µm 30 µm
Figure 7 Reduced fibrosis following follistatin gene therapy. Percent
fibrosis using picrosirius staining was quantified comparing pre- and
posttreatment muscle biopsies in high dose cohort. The error bars rep-
resent standard error of the mean. Posttreatment, we found that fibrosis
was reduced to 35% of baseline for Patient 05 and to 43% of baseline
for patient 06 (P < 0.017; mean percent fibrosis in Cohort 2 pretreat-
ment 33.14±4.47 versus posttreatment 19.28±1.73; one-way analysis
of variance).
Percent fibrosis
05. Pre 06. Pre05. Post 06. Post
© The American Society of Gene & Cell Therapy Follistatin Gene Therapy Trial in BMD
with the least o-target toxicity was an important step. e choice
was between two isoforms generated by alternative splicing. e
FS344 variant includes a C-terminal acidic region that undergoes
peptide cleavage to generate the serum circulating, nontissue
binding, FS315 isoform. is isoform avoids o-target eects
especially aecting sites within the pituitary-gonadal axis.56–59 Our
initial gene transfer experiments using AAV1.CMV.FS344 in the
mdx mouse demonstrated enhanced muscle mass and strength for
more than 2 years without adverse eects.18 We extended these
studies to nonhuman primates for up to 15 months without his-
tologic or functional adverse events to any key organ systems.19
e intramuscular injection of AAV1.CMV.FS344 to BMD
subjects in this clinical trial represents a successful proof-of-prin-
ciple study with an excellent safety prole that mirrored preclinical
ndings. e major clinical nding is the improvement in the dis-
tance walked on the 6MWT following injection of the quadriceps
muscles. ere was no apparent dierence in functional outcome
between low and high dose, with two of three patients improving
in each cohort. Impressively, two patients improved by over 100 m
in 6MWT. Two other patients improved, with increases of 58 m
and 29 m. Two patients failed to signicantly improve. We believe
that the greatest obstacle to gene expression-related improvement
was muscle brosis (Figures 3 and 4). Whereas in the normal mus-
cle of nonhuman primates, FS344 led to diuse muscle enhance-
ment,19 in BMD subjects with underlying widespread connective
tissue replacement of muscle, there were only focal areas of muscle
hypertrophy (Figure 5). us, future enrollment will benet from
pretreatment MRI assessment and MRI-guided gene transfer.
e extension of that nding is to avoid diuse brosis by early
intervention. Having said that, we did nd an anti-brotic eect
in endomysial brosis in regions of the biopsy where gene expres-
sion was apparent supported by ndings including a reduced
number of central nuclei, an increased in the number of muscle
bers, and a shi toward larger ber diameters and more uniform
ber distribution especially in high dose subjects. Overall these
ndings are consistent with follistatin-induced enhancement of
muscle dierentiation leading to more ecient regenerative activ-
ity.31 We also found reduced expression of miR-206 and muscle
expression of the specic follistatin isoform expressed following
AAV gene transfer. We did not nd a predictive correlation with
DMD gene mutations (Tabl e 1 ) or with dystrophin expression on
muscle biopsy prior to treatment (data not shown).
In preparation for this clinical trial, safety concerns were
raised regarding follistatin dysregulation of pituitary gonadotro-
pins, especially follicle-stimulating hormone (FSH) and lutein-
izing hormone (LH).57–59 FSH and LH are involved in control of
the reproductive function in vertebrates. In addition, follistatin
is found in gonads and pituitary tissues and autocrine/paracrine
eects on gonadotropins eects could be exerted by overexpres-
sion of follistatin in these tissues. Data generated from preclinical
studies in nonhuman primates showed no changes in FSH, LH,
testosterone or estrogen.19 is safety prole extended to the clini-
cal trial where we again saw no changes in gonadotropins, testos-
terone or estrogen levels following gene therapy (Supplementary
Figure S1). In addition, subjects in this clinical trial were closely
monitored for a wide range of toxicity in every organ system
and no abnormalities were encountered. Follistatin gene therapy
delivered by AAV1 under the control of a CMV promoter proved
to be exceptionally safe.
e safety ndings in combination with gene expression in
muscle, and functional improvement provide a rm foundation
for application of AAV1.FS344 gene delivery for other muscle dis-
eases. We have initiated a trial in sporadic inclusion body myositis
(sIBM). is is a challenging disease because of lack of treatment,
a long-term debilitating course, and an inammatory inltration
in muscle that responds poorly to immune suppression. e ability
of follistatin to target inammatory cells, promote muscle regen-
eration, and increase muscle ber size, provide signicant poten-
tial for a therapeutic eect in sIBM. We also have plans to extend
this trial of intramuscular AAV1.CMV.FS344 to DMD patients
changing the protocol to include a wider delivery of vector to mul-
tiple muscle groups. It is also noteworthy for future consideration
that dual vector delivery of AAV carrying FS344 in combination
with micro-dystrophin in mdx mice improved tetanic force and
provided full protection against eccentric contraction.60
In summary, the safety and ecacy as determined by the dis-
tance walked in the 6MWT, along with improved muscle histo-
pathology in a rst in human clinical trial of AAV1.CMV.FS344
warrants consideration for studies in other forms of muscular
dystrophy. is study also sets the stage for a pivotal clinical trial
for BMD patients.
Study subjects. Subject eligibility included proof of BMD mutation, knee
extensor weakness 2 standard deviation below normal,24 ambulatory, abil-
ity to cooperate for testing, willingness to practice contraception during
the study, and no evidence of cardiomyopathy, diabetes, or organ system
abnormalities of bone marrow, liver, or kidney. Human immunodeciency
virus infection, hepatitis A, B, or C, or known autoimmune diseases were
exclusion criteria. IRB approved consent forms were obtained by the
principal investigator (JRM) and signed by subjects. Consents included
approval for muscle biopsies performed under local anesthesia with inci-
sions made over the proximal vastus lateralis. A randomization table deter-
mined the side of the pretreatment biopsy. e postgene transfer biopsy
was done at 6 months postgene transfer to the same muscle of the opposite
extremity with particular eort to stay within the area of the gene injection
sites. Taking immunosuppressive drugs other than glucocorticoids during
the trial was prohibited.
e Institutional Review Board at Nationwide Children’s Hospital
approved this clinical trial. e protocol followed the Helsinki Declaration;
all patients gave their written informed consent and the trial was registered
at Clin.Trials.Gov.
Vector production
Purification and characterization. e AAV1 vector product was
produced using the AAV vector plasmid pAAV.CMV.FS344-Kan
(Supplementary Figure S8). It contains the human follistatin gene expres-
sion cassette anked by AAV2 inverted terminal repeat sequences (ITR).
It is this sequence that is encapsidated into AAV1 virions. e plasmid
was constructed by inserting the human follistatin cDNA sequence
(human cDNA, Genbank Accession # NM 013409) obtained from Origene
Technologies (Rockville, MD) into plasmid pAAV-MCS (Stratagene, La
Jolla, CA) using BamH I and Xho l restriction sites. e construct con-
tains the CMV immediate early promoter/enhancer and uses the β-globin
intron for high-level expression and the human growth hormone polyad-
enylation termination signal. Subsequently, the bla open reading frame
encoding ampicillin resistance was removed using BspH I digestion and
the kanamycin resistance gene (amino-glycoside 3-phosphotransferase II
Molecular erapy 7
© The American Society of Gene & Cell Therapy
Follistatin Gene Therapy Trial in BMD
gene) from Transposon Tn5 was PCR amplied with BspH I ends from
plasmid pSELECT-neo-mcs (InVivogen, San Diego, CA) and used to
replace the bla gene to yield the AAV vector plasmid pAAV.CMV.FS344-
Kan (5,347bp). e only viral sequences in this vector are the inverted
terminal repeats of AAV2, which are required for both viral DNA replica-
tion and packaging of the rAAV vector genome. All plasmids used in the
production process were produced by Aldevron under its GMP-S qual-
ity system and infrastructure utilizing the most salient features of cGMP
manufacturing; traceability, document control, and materials segregation.
rAAV1.CMV.FS344 was produced in the Nationwide Children’s Viral
Vector GMP Manufacturing Facility. Vector production followed previ-
ously published methods using plasmid DNA tri-transfection of HEK293
cells followed by iodixanol and anion exchange column chromatography
purication.25 Briey, cells were cultivated in ten-tray Corning Cell Stacks,
and all open manipulations were performed in class II biosafety cabinets
in an ISO Class 5 environment. e purication process was performed
in a closed system; where possible however, iodixanol gradient purica-
tion, an open step, was performed in an ISO Class 5 BSC. Purication con-
sisted of collecting the cells plus media and subjecting them to a single
pass microuidization at 1000 psig followed by clarication and tangential
ow ltration for volume reduction, iodixanol gradient purication and
anion exchange chromatography on the 40% iodixanol fraction. Aer
purication, the product was dialtered with nal formulation buer and
sterile ltered to yield the two Puried Bulks. Aer Puried Bulk testing,
the two Puried Bulks were pooled, diluted with sterile formulation buf-
fer (20 mmol/l Tris pH 8.0, 1 mmol/l MgCl2, 200 mmol/l NaCl, 0.001%
Pluronic F68) and a manual Final Fill was performed within a BSC in
the CMF Purication Room. Following Fill, the drug product underwent
release testing in anticipation of formal release by our Quality Assurance
Unit (QAU). Tests were performed on In-Process samples, the Puried
Bulk Drug Substance, and the Final Fill drug product along with stability
testing. Certicates of Stability and Analysis were submitted and approved
by the FDA. e DNase Resistant Particle titer (also referred to as vector
genomes (vg)) were determined for In-Process, Puried Bulk and Release
Testing samples using real-time quantitative PCR (qPCR) using serial dilu-
tions of a plasmid standard (pAAV.CMV.FS344-Kan) by the NCH-CMF
QC laboratory and CMV Forward Primer 5-TGG.AAA.TCC.CCG.TGA.
Functional measures. e primary functional outcome, the 6MWT was
performed at Nationwide Children’s Hospital by the same clinical evalu-
ators (L.P.L. and L.N.A.). e 6MWT was assessed at baseline prior to
the muscle biopsy. Single-day assessments were performed at 30 days,
60 days, 90 days, 6 months, and 1 year. Direct measure of maximum
voluntary isometric contraction of quadriceps muscles (knee extension)
served as a secondary outcome measure. ese outcome measures have
been previously described.29
Muscle biopsy analysis. Biopsies were obtained from the quadriceps mus-
cles, mounted in gum tragacanth and frozen in isopentane cooled in liquid
nitrogen. A standard battery of stains including H&E, modied Gomori
trichrome, and ATPase (pH 4.2, 4.6, and 9.4) was performed pre- and
posttreatment. H&E stained cross sections were used for ber size mea-
surements and internal nuclei determinations. Depending on the available
tissue size, 8–12 randomly selected areas were photographed at 20× and
stored. Fiber diameters were recorded with a calibrated micrometer, using
the AxioVision, 4.2 soware (Zeiss). Fiber size distribution histograms
were generated as number per mm2 area. ese same images were used
to identify the number of bers with either one or more central nuclei and
percent of bers with central nuclei. e amount of endomysial and peri-
mysial connective tissue was quantied in pre- and posttreatment biop-
sies using the Picro Sirius Red Stain Kit (Abcam ab150681). Twelve elds
were randomly selected in pre- and posttreatment biopsies and photo-
graphed at 20×; the level of brosis was analyzed with ImagePro soware.
Analysis was made using customs method with 2.5 minute counter stained
slides without color correction. Red area (as proportion of brotic area)
was expressed as percent of total area. e mean ± SE of the number of
images represented each biopsy. Pax7 positive satellite cells were identied
with mouse Pax7 IgG1 antibody (R&D systems) by immunohistochem-
istry protocol of Super Sensitive polymer-HRP detection kit using i6000
Automated Staining System from Biogenex. Briey, cryosections were
xed in 2% paraformaldehyde for 10 minutes at 4 °C and incubated in Pax7
antibody (1:100 dilutions) for 30 minutes aer blocking with peroxide and
Power Block for 10 minutes. Slides were washed ve times with IHC super-
sensitive wash buer. Finally, 3,3-Diaminobenzidine (DAB) was used as a
substrate and Mayor’s hematoxylin as a counterstain. Pax7 positive nuclei
counts were done using ImageScope soware (Apereo) and expressed as
number of Pax7 positive nuclei per muscle ber.
In pretreatment biopsies, immunohistochemistry was performed to
correlate dystrophin expression with outcome measures. e number
of dystrophin positive bers (NCL-Dys2, Novacastra Laboratories) and
quantication of dystrophin intensity were performed using Bioquant
image analysis soware (Nashville, TN).
RT-PCR was used to conrm expression of follistatin transcript
derived from the AAV.CMV.FS344 vector. RNA was isolated from
pre- and posttreatment biopsies and following cDNA conversion, a
vector specic PCR product was amplied using the following primers:
forward primer 5-CGAACATCGATTGAATTCCC-3 and reverse
primer 5-CTTGCTCAGTTCGGTCTT-3. To ensure specicity for
amplication of vector derived transcript, the forward primer was
designed to be complementary to an unspliced and transcribed region in
the distal 3 region of the CMV promoter with the reverse primer binding
to the follistatin transgene.
Quantitative PCR to detect genome copy number. Taqman qPCR was used
to quantify the number of vector genome copies compared to baseline biop-
sies as previously described.26,27 A vector specic primer probe was used to
determine the copy number, reported as vector genomes per microgram of
genomic DNA. e primer sets amplied a unique sequence of the CMV pro-
moter within the CMV.FS cassette: 5-TGGAAATCCCCGTGAGTCAA-3;
a CMV reverse primer, 5-CATGGTGATGCGGTTTTGG-3; and CMV
Identification of muscle specific microRNA expression. Total RNA was
isolated from the specimens using mirVana miRNA isolation kit (Life
Technologies). Reverse transcription was performed by using Taqman
microRNA reverse transcription kit (Applied Biosystems). Quantitative
reverse transcription-polymerase chain reaction (qRT-PCR) for miR-1,
miR-206, miR-133a, and U6 snRNA was performed using RT kits from
Life Technologies specic for each miR.
e catalog numbers for each as follows, miR-1: 4427975, ID 002222,
miR-206: 4427975, ID 000510, miR-29c: 4427975, ID000587, U6:
4427975, ID001973.
Each miRNA expression was normalized to U6 snRNA expression.
Expression data is shown as means of relative expression values obtained
from three samples and normalized to normal control levels (set at 1).
Standard error of means and presented in a graph format.
ELISpot analysis. ELISpot (enzyme-linked immunospot) assays
were performed on fresh PBMCs, which were added at a concentration
of 2×105/well in duplicate wells of a 96-well at-bottom membrane-plate
(Millipore, Billerica, MA). ree peptide pools were used for the AAV1
capsid protein (Genemed Synthesis), containing 34–36 peptides, each
18 amino acids long and overlapping by 11 residues. Two peptide pools
encompassing the follistatin protein (Genemed Synthesis) were used as
previously described,18 Concanavalin A (Sigma) served as a positive con-
trol, and 0.25% DMSO as a negative control. Peptides were added directly
to the wells at a nal concentration of 1 µg/ml in 200 µl of AIM-HS (Aim-V
lymphocyte media (Invitrogen) supplemented with 2% human AB serum
© The American Society of Gene & Cell Therapy Follistatin Gene Therapy Trial in BMD
(Gemini-BioScience BLCL medium) RPMI 1640 (Gibco) supplemented
with 10% fetal bovine serum (Gibco) and Pen Strep (Gibco)). Human IFN-γ
ELISpot kits were purchased from U-CyTech (Utrecht, Netherlands). Aer
the addition of PBMCs and peptides, the plates were incubated at 37 °C
for 48 hours and then developed according to the manufacturer’s protocol.
IFN-γ-spot formation was counted using a Cellular Technologies Limited
Systems analyzer (CTL, Cleveland, OH).
Anti-AAV neutralizing antibody titers. e assay is based on the ability
of neutralizing antibody (Nab) in serum to block target cell transduction
with a B-gal reporter vector stock. C12 rep expressing HeLa cells (Viral
Vector Core, Nationwide Children’s Hospital) were plated in a 96-well plate
(Corning) at a concentration of 5e4 cells/well, Plates were incubated at 37
°C with 5% CO2. e following day, an aliquot of patient serum was heat
inactivated for 30 minutes at 56 °C. Serum was diluted in duplicate twofold
with DMEM in a 96-well plate so that the plate contained 1:50–1:1,638,400
dilutions. 5e7 DRP/ml AAV1.CMV.βgal virus was added to the serially
diluted wells in a volume of 25 µl. For the assay cuto, 25 µl of 5e7, 1e7,
and 5e6 DRP/ml were added to other wells containing 1:50 diluted naïve
serum e 96-well plates were then rocked for 2–5 minutes, and incubated
for 1 hour at 37 °C. Media was then removed and all 50 µl of the diluted
serum/AAV1 complexes were added to the corresponding well containing
C12 cells. 50 µl of the Ad5 (MOI = 250) were added to the diluted serum
Aer overnight incubation at 37 °C, the media was replaced with 10%
FBS DMEM media the media was removed aer 36 hours. of incubation
and gently washed with 200 µl/well of PBS (Invitrogen). 100 µl/well of Pierce
β-gal Assay Reagent (ermo Scientic) were added and incubated for 30
minutes at 37 °C. e plates were then read at 405nm on a SPECTRA max
M2 plate reader (Molecular Devices). e 5e6 DRP/ml positive control was
the assay cuto, which represents an equivalent of 10% infection and 90%
neutralization. e farthest serum dilution producing an average absorbance
at 405nm that was less than the average absorbance of the 5e6 DRP/ml
positive control was considered the anti-AAV1 titer.
Anti-follistatin antibody titers. An ELISA (Enzyme-Linked Immunosorbent
Assay) was performed to measure the level of circulating anti-follistatin anti-
body in plasma. Briey, Immulon-4 96-well plates (ISC BioExpress) were
coated with 100 µl of human follistatin protein in carbonate buer (pH 9.4;
Pierce) per well. Plates were sealed overnight at 4 °C. Plates were blocked
with 280 µl per well of a 5% nonfat dry milk and 1% normal goat serum
(Invitrogen) in PBS for 3 hours at 25 °C. Patient plasma was diluted at a 1:50
ratio in solution identical to the blocking solution and 100 µl added in dupli-
cate to both wells coated with follistatin in carbonate buer and wells coated
with carbonate buer alone. Plates were incubated at 25 °C for 1 hour before
being washed ve times with 280 µl of PBS-T (0.05% Tween). Blocking solu-
tion was used again to dilute the secondary antibody, goat anti-human IgG-
HRP (Sigma) at a 1:10,000 dilution. Wells received 250 µl of the secondary
antibody and were incubated at 25 °C for 30 minutes before being washed
ve times and blotted dry. Tetramethybenzidine (TMB; 100 µl/well; Pierce)
was added and incubated at 25 °C for 10 minutes in the dark, before the addi-
tion of 100 µl of 1 N H2SO4 (Acros Organics) to stop the reaction. e absor-
bance at 450 Å was measured using a Wallace 1420-050 Multilabel Counter
(Perkin Elmer). Samples were considered positive if the absorbance at 450 Å
average of the antigen-coated wells was three times greater than wells coated
with carbonate buer alone.
Muscle imaging. Muscle MRI was performed using T1 weighted spin echo
on a 3.0 Tesla GE Signa Excite (General Electric Healthcare; Milwaukee,
WI). Noncontrast enhanced images obtained from both legs were col-
lected at baseline and 6 months postgene therapy treatment for all six
subjects. Axial T1-weighted images of the lower extremities to the knees
were obtained to study pelvic and thigh musculature. A body coil was
used for obtaining T1 spin echo pulse sequences (repetition time (TR) 650
microseconds; echo time (TE) 15 microseconds) with a 256×256 matrix
and a slice thickness of 10mm each with no gap between slices. A eld of
view (FOV) of 480mm was used and a total of 48 slices for each leg was
obtained. A retrospective analysis of the images was performed by apply-
ing a semi-quantitative method for grading the degree of individual muscle
involvement.61–63 Grading of muscles was based on the following scoring
• Stage 0: Normal appearance
• Stage 1: Scattered small areas of increased intensity
• Stage 2a: Numerous discrete areas of increased intensity less than
30% of the volume of the muscle
• Stage 2b: Numerous discrete areas of increased intensity with early
conuence, 30–60% of the volume of the muscle
• Stage 3: Washed-out appearance due to conuent areas increased
intensity with muscle still present at the periphery
• Stage 4: End-stage appearance, muscle entirely replaced by areas of
increased intensity
Analysis of degree of muscle involvement on MRI using the above
described scoring system was performed by two independent observers
(S.A-Z. and A.G.) and a consensus on the scoring was reached for
all muscle groups in all six subjects. Individual muscles were graded
separately with the exception of the vastus lateralis and intermedius that
were graded as one muscle due to poorly dierentiated boundaries.
Statistical analyses. GraphPad Prism soware (La Jolla, CA) was used
for all statistical analyses. For all comparisons, two-tailed Student’s t-test
was used or where appropriate one-way analysis of variance was applied.
Avalue of P < 0.05 was considered statistically signicant.
Figure S1. Hormonal profile for follistatin-treated patients.
Figure S2. Muscle fiber size distribution histograms from pre and
posttreatment biopsies.
Figure S3. Picrosirius red collagen staining of muscle pre- and postfol-
listatin treatment.
Figure S4. Follistatin gene therapy and central nucleation.
Figure S5. Pre-and posttreatment RT-PCR on muscle biopsies.
Figure S6. Pax7 positive nuclei per muscle fiber in pre- and posttreat-
ment biopsies for Patients 01, 02, 05, and 06.
Figure S7. miR-206, miR-1, and miR29c levels in pre- and posttreat-
ment muscle biopsies for Patients 01, 02, 05, and 06.
Figure S8. AAV.CMV.FS344-Kan plasmid used for vector production.
Table S1. Follistatin gene therapy adverse events.
Table S2. Follistatin DNA copy number.
The Parent Project Muscular Dystrophy supported the Clinical Trial.
Staff for this trial and some of the materials and supplies were supplied
by the Senator Paul D Wellstone Muscular Dystrophy Research Center,
NICHD, NIH, Bethesda, MD #5U54HD066409-05. Jesse’s Journey sup-
ported some of the participating staff. The Myositis Association (TMA)
made helped bring this trial to the clinic by supporting the preclinical
studies. The authors declare no conflict of interest.
BKK had intellectual property filed through Nationwide Children’s
Hospital and an equity interest related to work that is licensed to Milo
Biotechnology. BKK also serves as a paid consultant for Milo. The rela-
tionships are managed through a conflict management plan.
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... Pre-clinical trials of FST344 in cynomolgus monkeys demonstrated an improvement in muscle mass and strength [64]. Likewise, the patients with Becker muscular dystrophy and sIBM improved walking speed following the FST344 gene transfer [65,66]. However, further trials are underway, and the results are under evaluation. ...
... Transgenic expression of follistatin in mdx mice, a model for Duchenne muscular dystrophy, showed amelioration of dystrophic pathology and an increase in skeletal muscle mass [198]. Interestingly, in a gene therapy trial, Mendell et al. demonstrated beneficial effects of follistatin in direct delivery into intramuscular quadriceps in patients suffering from Becker Muscular Dystrophy without any apparent side effects [199]. Follistatin was identified as a direct target of the testosterone effect during its promyogenic action in both mouse models [200] and cell culture studies [201]. ...
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Obesity is characterized by an excessive accumulation of fat leading to a plethora of medical complications, including coronary artery disease, hypertension, type 2 diabetes mellitus or impaired glucose tolerance and dyslipidemia. Formerly, several physiological roles of organokines, including adipokines, hepatokines, myokines and gut hormones have been described in obesity, especially in the regulation of glucose and lipid metabolism, insulin sensitivity, oxidative stress, and low-grade inflammation. The canonical effect of these biologically active peptides and proteins may serve as an intermediate regulatory level that connects the central nervous system and the endocrine, autocrine, and paracrine actions of organs responsible for metabolic and inflammatory processes. Better understanding of the function of this delicately tuned network may provide an explanation for the wide range of obesity phenotypes with remarkable inter-individual differences regarding comorbidities and therapeutic responses. The aim of this review is to demonstrate the role of organokines in the lipid and glucose metabolism focusing on the obese non-diabetic subgroup. We also discuss the latest findings about sarcopenic obesity, which has recently become one of the most relevant metabolic disturbances in the aging population.
... Follistatin is also capable of inhibiting myostatin, a protein thought to be responsible for muscular dystrophy, with follistatin administration being of particular interest in this as means for enhancing follistatin levels in the human body [70]. One example is the phase I/IIA clinical trial for the use of a follistatin construct introduced via an adeno-associated virus for the treatment of Becker muscular dystrophy [108]. Recombinant follistatin has also been developed, such as ACE-083, although results on its efficacy against facioscapulohumeral muscular dystrophy conditions have not been promising [107,109]. ...
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Advances in technology and biomedical knowledge have led to the effective diagnosis and treatment of an increasing number of rare diseases. Pulmonary arterial hypertension (PAH) is a rare disorder of the pulmonary vasculature that is associated with high mortality and morbidity rates. Although significant progress has been made in understanding PAH and its diagnosis and treatment, numerous unanswered questions remain regarding pulmonary vascular remodeling, a major factor contributing to the increase in pulmonary arterial pressure. Here, we discuss the role of activins and inhibins, both of which belong to the TGF-β superfamily, in PAH development. We examine how these relate to signaling pathways implicated in PAH pathogenesis. Furthermore, we discuss how activin/inhibin-targeting drugs, particularly sotatercep, affect pathophysiology, as these target the afore-mentioned specific pathway. We highlight activin/inhibin signaling as a critical mediator of PAH development that is to be targeted for therapeutic gain, potentially improving patient outcomes in the future.
... huFollistatin344, which is an intramuscular transfer of the human follistatin, a protein that binds to myostatin and inhibits its activity, was assessed for efficacy in a phase 1-2 clinical study (NCT02354781) in Becker muscular dystrophy (BMD) patients. Results showed an excellent safety profile that mirrored preclinical findings, as well as an improvement in the distance walked following injection of the quadriceps muscles (Mendell et al., 2015). Limitation to this application are that its use on DMD patients is still to fully investigate, since pathogenesis are different and symptoms of BMD are lighter. ...
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Introduction: This systematic review analyzes the state-of-art repurposing of the drug tamoxifen (TAM) in the treatment of Duchenne Muscular Dystrophy (DMD), including its mechanism of action, toxicological findings, and past and ongoing clinical trials. A parallel aim of this work was to explore whether evidence exists to support further funding of investigation on TAM treatment for DMD patients with a pivotal trial in young patients. Bringing evidence and answering the scientific question of whether this treatment could improve the quality-of-life of DMD patients is needed to establish guidelines and accelerate access to promising therapies for DMD patients. Methods: The search was conducted in January 2022 utilizing PubMed. All MeSH terms for “Duchenne Muscular Dystrophy” and “tamoxifen” were used. The inclusion and exclusion criteria were defined according to the PICOS framework. Results: The included publications all explored the use of TAM with promising outcomes in muscular strength recovery and a decrease in pathology biomarkers. Two reviews recognize TAM as a potential treatment for DMD patients and state that drug repurposing plays a crucial role in the quest for a drug candidate to treat this rare disease. Conclusion: According to available data, TAM shows promise as a treatment for DMD, both pharmacologically and clinically. However, published data to date are insufficient to definitively conclude the beneficial effect of TAM on quality-of-life and ultimately survival, particularly in the youngest patients diagnosed with DMD.
... Becker muscular dystrophy (BMD) is a type of muscle dystrophin deficiency caused by a mutation in the DMD gene. Even though BMD has a less severe clinical history than Duchenne muscular dystrophy (DMD), the phenotypic heterogeneity is great [9]. Facial muscles, shoulder girdles, and upper arms are affected by facioscapulohumeral muscular dystrophy (FSHD), which is followed by trunk and distal lower extremities, and more proximal muscular weakening later in the disease course. ...
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Muscular dystrophies (MDs) are a category of hereditary illnesses characterized by the gradual malfunction and/or weakening of the skeletal muscles. This disease of the muscles also results in hypotonia and joint contracture, along with raised serum creatine kinase (CK) levels. To prevent complications, continuous physiotherapy is advised for children with muscular dystrophy, which is even asked to perform at home as a home exercise program (HEP). As a result, the home exercise program (HEP) is critical in maintaining the optimal health of children with Duchenne muscular dystrophy (DMD). The present coronavirus (COVID-19) pandemic has adversely affected these children as there was very little scope to get direct help from a physiotherapist. Meanwhile, the home program was continued by many to compensate for the direct benefit. However, because of the lack of specific guidelines and structured methodology to follow for a home program, there was a deterioration in the health status of many children. There is a need to understand how the children are getting affected and the way the home program can be refined to help needy children with muscular dystrophy. Our scoping review aims to identify the present home program patterns being followed for children with DMD and their scope for refinement. The data were collected from electronic databases including PubMed, ProQuest, Cochrane, and Web of Science. We searched four electronic databases until September 2021. We included the published case studies, observational and experimental studies that described the positive impact of home exercise programs, and the methodology they followed as an alternative to institution-based physiotherapy. One hundred thirty-eight titles were screened, and 58 met the inclusion criteria. Along with regular physiotherapy, the incorporation of HEP helped in early complication prevention in patients with muscular dystrophy. The HEP was found to be a successful adjunct in the COVID-19 scenario. This review presents different therapeutic measures that can be taken for the prevention of complications in patients with MD and how the HEP plays an important role in removing the gaps on how HEP is beneficial in the COVID-19 scenario and a scope to refine the present methodologies for more accurate management.
... La découverte du rôle de la myostatine sur la trophicité musculaire a également inspiré plusieurs essais thérapeutiques avec des inhibiteurs de la myostatine. Il faut rappeler que la première thérapie génique chez l'homme ayant conduit à une amélioration de la force musculaire a été menée chez les patients atteints de la dystrophie musculaire de Becker avec un inhibiteur direct de la myostatine, la follistatine [5]. L'expression du gène de la myostatine dépend de nombreux facteurs. ...
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In order to explain muscle hypertrophy in a patient complaining of muscular symptoms one must take into account the patient’s daily professional or leisure physical activity. If muscle hypertrophy cannot be attributed to active lifestyle, an underlying neuromuscular pathology should be suspected. The check-up includes a meticulous neurological examination (looking for myotonia, rippling, myoclonus, etc.), a CK level dosage, an electromyography (for myotonic discharges), and, if necessary, it should be completed by muscle biopsy and molecular examinations. Several diagnostic hypotheses should be considered. Most are related to a genetic defect leading to dysfunction of the excitation-contraction coupling in the muscle. The diagnosis of a myotonic syndrome is the most common but caveolinopathy 3, RYR1-related myopathy or Brody’s disease are also possible.
Muscular dystrophies are a group of geneticGenetics disorders characterized by varying degrees of progressive muscle weakness and degeneration. They are clinically and genetically heterogeneous but share the common histological features of dystrophic muscle. There is currently no cure for muscular dystrophies, which is of particular concern for the more disabling and/or lethal forms of the disease. Through the years, several therapies have encouragingly been developed for muscular dystrophies and include geneticGenetics, cellular, and pharmacological approaches. In this chapter, we undertake a comprehensive exploration of muscular dystrophy therapeutics under current development. Our review includes antisense therapy, CRISPR, gene replacement, cell therapy, nonsense suppression, and disease-modifying small molecule compounds.
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Tumors invade the surrounding tissues to progress, but the heterogeneity of cell types at the tumor-stroma interface and the complexity of their potential interactions hampered mechanistic insight required for efficient therapeutic targeting. Here, combining single-cell and spatial transcriptomics on human basal cell carcinomas, we define the cellular contributors of tumor progression. In the invasive niche, tumor cells exhibit a collective migration phenotype, characterized by the expression of cell-cell junction complexes. In physical proximity, we identify cancer-associated fibroblasts with extracellular matrix-remodeling features. Tumor cells strongly express the cytokine Activin A, and increased Activin A-induced gene signature is found in adjacent cancer-associated fibroblast subpopulations. Altogether, our data identify the cell populations and their transcriptional reprogramming contributing to the spatial organization of the basal cell carcinoma invasive niche. They also demonstrate the power of integrated spatial and single-cell multi-omics to decipher cancer-specific invasive properties and develop targeted therapies.
Duchenne muscular dystrophy is an X-linked disease caused by the absence of functional dystrophin in the muscle cells. Major advances have led to the development of gene therapies, tools that induce exon skipping, and other therapeutic approaches, including treatments targeting molecular pathways downstream of the absence of functional dystrophin. However, glucocorticoids remain the only treatment unequivocally shown to slow disease progression, despite the adverse effects associated with their long-term use. Besides glucocorticoids, which are standard care, five compounds have received regulatory approval in some but not all jurisdictions, with further efficacy results being awaited. Several compounds with promising results in early-phase clinical trials have not met their efficacy endpoints in late-phase trials, but the clinical development of many other compounds is ongoing. The current landscape is complicated by the number of compounds in various stages of development, their various mechanisms of action, and their genotype-specific applicability. The difficulties of clinical development that arise from both the rarity and variability of Duchenne muscular dystrophy might be overcome in the future by use of sensitive biomarkers, natural history data, and ameliorated trial designs.
Duchenne muscular dystrophy (DMD) is a severe, progressive, genetic muscle wasting disorder arising from the absence of the membrane stabilizing protein, dystrophin, that renders muscle fibers susceptible to damage and degeneration. Since the discovery of the dystrophin gene, research efforts have focused on the development of regenerative gene- and cell-based therapies for DMD, although many obstacles need to be overcome before they can be considered for clinical application. The development of adjunct therapies that can slow the pathologic progression, preserve muscle mass, enhance muscle regeneration, and promote muscle growth, is therefore essential. Rehabilitation through physical exercise or muscle contraction protocols may help attenuate muscle weakness and dysfunction in DMD, with evidence supporting rehabilitation as an adjunct treatment to gene- and cell-mediated therapies. This chapter summarizes the current state of research for DMD therapy and explores the potential for combined regenerative and rehabilitation therapies to improve outcomes for DMD patients.KeywordsDuchenne muscular dystrophyGene therapyCell therapyRehabilitationExercise
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Follistatin (FST) is a member of the TGFβ family and is a secreted glycoprotein that antagonizes many members of the family including Activin A, GDF11 and myostatin. The objective of this study was to explore the use of an engineered Follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N-terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.
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Introduction: Duchenne muscular dystrophy (DMD) subjects ≥5 years with nonsense mutations were followed for 48 weeks in a multicenter, randomized, double-blind, placebo-controlled trial of ataluren. Placebo arm data (N = 57) provided insight into the natural history of the 6-minute walk test (6MWT) and other endpoints. Methods: Evaluations performed every 6 weeks included the 6-minute walk distance (6MWD), timed function tests (TFTs), and quantitative strength using hand-held myometry. Results: Baseline age (≥7 years), 6MWD, and selected TFT performance are strong predictors of decline in ambulation (Δ6MWD) and time to 10% worsening in 6MWD. A baseline 6MWD of <350 meters was associated with greater functional decline, and loss of ambulation was only seen in those with baseline 6MWD <325 meters. Only 1 of 42 (2.3%) subjects able to stand from supine lost ambulation. Conclusion: Findings confirm the clinical meaningfulness of the 6MWD as the most accepted primary clinical endpoint in ambulatory DMD trials.
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microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass in vivo, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 "sponge," featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype - an important consideration in relation to the development of therapeutics designed to manipulate microRNA activity in musculature.
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Pharmacologic strategies have provided modest improvement in the devastating muscle-wasting disease, Duchenne muscular dystrophy (DMD). Pre-clinical gene therapy studies have shown promise in the mdx mouse model; however studies conducted after disease onset fall short of fully correcting muscle strength or protecting against contraction-induced injury. Here we examine the treatment effect on muscle physiology in aged dystrophic mice with significant disease pathology by combining two promising therapies: micro-dystrophin gene replacement and muscle enhancement with follistatin, a potent myostatin inhibitor. Individual treatment with micro-dystrophin and follistatin demonstrated marked improvement in mdx mice, but were insufficient to fully restore muscle strength and response to injury to wild-type levels. Strikingly, when combined, micro-dystrophin/follistatin treatment restored force generation and conferred resistance to contraction-induced injury in aged mdx mice. Pre-clinical studies with miniature dystrophins have failed to demonstrate full correction of the physiological defects seen in mdx mice. Importantly, the addition of a muscle enhancement strategy with delivery of follistatin in combination with micro-dystrophin gene therapy completely restored resistance to eccentric contraction induced injury and improved force. Eccentric contraction induced injury is a pre-clinical parameter relevant to the exercise induced injury that occurs in DMD patients and herein we demonstrate compelling evidence for the therapeutic potential of micro-dystrophin/follistatin combinatorial therapy.
Objective: Patients with Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy lack neuronal nitric oxide synthase (nNOS). nNOS mediates physiological sympatholysis, thus ensuring adequate blood supply to working muscle. In mice lacking dystrophin, restoration of nNOS effects by a phosphodiesterase 5 (PDE5) inhibitor (sildenafil) improves skeletal and cardiac muscle performance. Sildenafil also improves blood flow in patients with BMD. We therefore hypothesized that sildenafil would improve blood flow, maximal work capacity, and heart function in patients with BMD. Methods: A randomized, double-blind, placebo-controlled crossover design with two 4-week periods of treatment, separated by 2-week washout was used. We assessed brachial artery blood flow during maximal handgrip exercise, 6-minute walk test, maximal oxidative capacity, and life quality; cardiac function was evaluated by magnetic resonance imaging (MRI) at rest and during maximal handgrip exercise. Muscle nNOS and PDE5 were tested with Western blotting in 5 patients. Results: Sixteen patients completed all skeletal muscle evaluations, and 13 completed the cardiac MRI investigations. Sildenafil had no effect on any of the outcome parameters. No serious adverse effects were recorded. PDE5 and nNOS were deficient in 5 of 5 biopsies. Interpretation: Despite positive evidence from animal models of dystrophinopathy and physiological findings in patients with BMD, this double-blind, placebo-controlled clinical study showed no effect of sildenafil on blood flow, maximal work capacity, and heart function in adults with BMD. This discrepancy may be explained by a significant downregulation of PDE5 in muscle.
Objective: Duchenne and Becker muscular dystrophies (DBMD) are allelic disorders caused by mutations in dystrophin. Adults with DBMD develop life-threatening cardiomyopathy. Inhibition of phosphodiesterase 5 (PDE5) improves cardiac function in mouse models of DBMD. To determine whether the PDE5-inhibitor sildenafil benefits human dystrophinopathy, we conducted a randomized, double-blind, placebo-controlled trial (, number NCT01168908). Methods: Adults with DBMD and cardiomyopathy (ejection fraction ≤ 50%) were randomized to receive sildenafil (20mg 3× daily) or placebo for 6 months. All subjects received an additional 6 months of open-label sildenafil. The primary endpoint was change in left ventricular end-systolic volume (LVESV) on cardiac magnetic resonance imaging. Secondary cardiac endpoints, skeletal muscle function, and quality of life were also assessed. Results: An interim analysis (performed after 15 subjects completed the blinded phase) revealed that 29% (4 of 14) of subjects had a ≥10% increase in LVESV after 6 months of sildenafil compared to 13% (1 of 8) of subjects receiving placebo. Subjects with LVESV > 120ml at baseline were more likely to worsen at 12 months regardless of treatment assignment (p = 0.035). Due to the higher number of subjects worsening on sildenafil, the data and safety monitoring board recommended early termination of the study. There were no statistically significant differences in outcome measures between treatment arms. Interpretation: Due to the small sample size, comparisons between groups must be interpreted with caution. However, this trial suggests that sildenafil is unlikely to improve cardiac function in adults with DBMD.
In prior open-label studies, eteplirsen, a phosphorodiamidate morpholino oligomer (PMO), enabled dystrophin production in Duchenne muscular dystrophy (DMD) with genetic mutations amenable to skipping exon 51. The present study used a double-blind placebo-controlled protocol to test eteplirsen's ability to induce dystrophin production and improve distance walked on the 6-minute walk test (6MWT). DMD boys aged 7 to 13, with confirmed deletions correctable by skipping exon 51 and ability to walk 200-400 meters on 6-minute walk test (6MWT) were randomized to weekly intravenous infusions of 30 or 50 mg/kg/week eteplirsen or placebo for 24 weeks (n=4/group). Placebo-patients switched to 30 or 50 mg/kg eteplirsen (n=2/group) at Week 25; treatment was open-label thereafter. All patients had muscle biopsies at baseline and week 48. Efficacy included dystrophin-positive fibers and distance walked on the 6MWT. At Week 24, the 30-mg/kg eteplirsen patients were biopsied and percentage of dystrophin-positive fibers increased to 23% of normal; no increases were detected in placebo-treated patients (p≤0.002). Even greater increases occurred at week 48 (52% and 43% in the 30 and 50 mg/kg cohorts, respectively) suggesting dystrophin increases with longer treatment. Restoration of functional dystrophin was confirmed by detection of sarcoglycans and nNOS at the sarcolemma. Ambulation-evaluable eteplirsen-treated patients experienced a 67.3-meter benefit compared to placebo/delayed patients (p≤0.001). Eteplirsen restored dystrophin in the 30 and 50 mg/kg/wk cohorts, and in subsequently treated, placebo-controlled subjects. Duration, more than dose, accounted for dystrophin production, also resulting in ambulation stability. No severe adverse events were encountered. ANN NEUROL 2013. © 2013 American Neurological Association.
Becker muscular dystrophy (BMD) is characterised by broad clinical variability. Ongoing studies exploring dystrophin restoration in Duchenne muscular dystrophy ask for better understanding of the relation between dystrophin levels and disease severity. We studied this relation in BMD patients with varying mutations, including a large subset with an exon 45-47 deletion. Dystrophin was quantified by western blot analyses in a fresh muscle biopsy of the anterior tibial muscle. Disease severity was assessed using quantitative muscle strength measurements and functional disability scoring. MRI of the leg was performed in a subgroup to detect fatty infiltration. 33 BMD patients participated. No linear relation was found between dystrophin levels (range 3%-78%) and muscle strength or age at different disease milestones, in both the whole group and the subgroup of exon 45-47 deleted patients. However, patients with less than 10% dystrophin all showed a severe disease course. No relation was found between disease severity and age when analysing the whole group. By contrast, in the exon 45-47 deleted subgroup, muscle strength and levels of fatty infiltration were significantly correlated with patients' age. Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45-47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.
Introduction: Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up-regulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. Methods: We examined the histopathological stages, Pax7 SC content, and muscle-specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis. Results: Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in the fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206. Conclusions: These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.