Gene Therapy (2010) 17, 432–438
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SHORT COMMUNICATION
Dystrophin restoration in skeletal, heart and skin
arrector pili smooth muscle of mdx mice by ZM2
NP–AON complexes
A Ferlini1, P Sabatelli1,2, M Fabris1, E Bassi1, S Falzarano1, G Vattemi3, D Perrone4, F Gualandi1,
NM Maraldi5, L Merlini1, K Sparnacci6, M Laus6, A Caputo7, P Bonaldo7, P Braghetta7 and P Rimessi1
1
Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy; 2IGM-CNR,
Unit of Bologna c/o IOR, Bologna, Italy; 3Department of Neurological Sciences and Vision, Section of Clinical Neurology, University of
Verona, Verona, Italy; 4Department of Biology and Evolution, University of Ferrara, Ferrara, Italy; 5Department of Human Anatomical
Sciences, University of Bologna and Laboratory of Cell Biology, IOR, Bologna, Italy; 6Department of Environmental and Life Sciences
INSTM, University of Eastern Piedmont, Alessandria, Italy and 7Department of Histology, Microbiology, and Medical Biotechnology,
University of Padua, Padua, Italy
Potentially viable therapeutic approaches for Duchenne
muscular dystrophy (DMD) are now within reach. Indeed,
clinical trials are currently under way. Two crucial aspects
still need to be addressed: maximizing therapeutic efficacy
and identifying appropriate and sensible outcome measures.
Nevertheless, the end point of these trials remains painful
muscle biopsy to show and quantify protein restoration in
treated boys. In this study we show that PMMA/N-isopropilacrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey
antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2–AON complex restored dystrophin
protein synthesis in both skeletal and cardiac muscles of mdx
mice, allowing protein localization in up to 40% of muscle
fibers. The mdx exon 23 skipping level was up to 20%, as
measured by the RealTime assay, and dystrophin restoration
was confirmed by both reverse transcription-PCR and
western blotting. Furthermore, we verified that dystrophin
restoration also occurs in the smooth muscle cells of the
dorsal skin arrector pili, an easily accessible histological
structure, in ZM2–AON-treated mdx mice, with respect to
untreated animals. This finding reveals arrector pili smooth
muscle to be an appealing biomarker candidate and a novel
low-invasive treatment end point. Furthermore, this marker
would also be suitable for subsequent monitoring of the
therapeutic effects in DMD patients. In addition, we
demonstrate herein the expression of other sarcolemma
proteins such as a-, b-, g- and d-sarcoglycans in the human
skin arrector pili smooth muscle, thereby showing the
potential of this muscle as a biomarker for other muscular
dystrophies currently or soon to be the object of clinical trials.
Gene Therapy (2010) 17, 432–438; doi:10.1038/gt.2009.145;
published online 12 November 2009
Keywords: dystrophin; antisense; nanoparticles; arrector pili; smooth muscle
Duchenne muscular dystrophy (DMD) is an inherited
X-linked degenerative muscular disorder predominantly
caused by frame-disrupting mutations arising from gross
rearrangements in the dystrophin gene.1 DMD patients
are affected by both severe skeletal muscle wasting and
dilated cardiomyopathy. The milder allelic form of the
disease, Becker muscular dystrophy, is due to in-frame
mutations that preserve a shortened but functional
protein.
Recently, novel therapeutic approaches for DMD have
emerged, some of which have thus far only been tested in
animal models. Others, however, such as drugs which
induce stop-codon reversion2 (PTC124) and antisense
oligoribonucleotide (AON)-mediated exon skipping,3–5
Correspondence: Professor A Ferlini, Department of Experimental
and Diagnostic Medicine, Section of Medical Genetics, University
of Ferrara, Via Fossato di Mortara, 74, Ferrara, FE 44100, Italy.
E-mail: [email protected]
Received 27 July 2009; revised 23 September 2009; accepted 25
September 2009; published online 12 November 2009
are already undergoing clinical trials.6 These novel therapeutic options for DMD represent the first step toward a
possible cure for this devastating disorder. However,
several major obstacles, namely optimization of nontoxic
effective doses, improvement of the delivery system,
distribution to all affected tissues, achievement of a
sustainable therapeutic effect and development of an
administration strategy suitable for lifelong treatment,7–10
still remain to be overcome.
We previously showed that inert PMMA T1 nanoparticles (NPs) conjugated with AON are capable of
inducing dystrophin restoration in body-wide muscles
in the mdx animal model.11 Although encouraging in
terms of dystrophin-positive fiber percentage, indeed we
obtained a functional effect with one-eightieth of the
normal AON dose regimen, the efficiency of this novel
compound was relatively unsatisfactory. Therefore, to
obtain a more efficient compound, we designed and
prepared a novel type of cationic core-shell NP (termed
ZM2), made up of a predominantly PMMA core and a
random copolymer shell consisting of units derived from
Nanoparticle–AON dystrophin rescue
A Ferlini et al
N-isopropil-acrylamide+ (NIPAM) and reactive methacrylate-bearing cationic groups (Supplementary Figure
S1, a and b). These novel NPs were found to be nontoxic
in vitro, even following repeated administrations. The
cytotoxicity of ZM2 NP was assayed in HeLa cells
following incubation with increasing amounts of NPs
(see Supplementary Materials and methods and Supplementary Figure S2), and no significant reduction in cell
viability was observed up to 1 mg ml1, as compared
with untreated cells (P40.05).
Moreover, adsorption of the M23D (+0718) AON 20 O-methyl full-length phosphorothioate (20 OMePS)11 onto
ZM2 NPs proved to be a highly reproducible process at a
loading value of 90 mg of AON per mg of NPs (see
Supplementary Materials and methods, and Result).
We initially divided 6-week-old mdx mice into two
groups: group 1 (four animals) received 225 mg intraperitoneal injections of naked M23D AON (naked AON)
and group 2 (four animals) received intraperitoneal
injections of 225 mg of M23D AON conjugated with
2.5 mg of ZM2 NPs. A further four mdx mice were
injected with 2.5 mg of ZM2 NPs dissolved in sterile
unpreserved saline solution (0.9% sodium chloride); four
non-injected mdx mice were used as controls. The
animals were injected weekly for 7 weeks (7.5 mg kg1
per injection) and killed 1 week after the final administration. The total amount of M23D AON received by each
animal was 52.5 mg kg1 (1575 mg per animal).
Immunohistochemical analysis subsequently demonstrated that the ZM2–AON complex induces dystrophin
restoration very efficiently in the skeletal muscles of the
quadriceps, gastrocnemius and diaphragm as well as in
the cardiac muscle (Figure 1). Seven-micron-thick frozen
transverse sections of skeletal and cardiac muscles from
untreated, naked AON-treated, ZM2–AON-treated mdx
mice and wild-type mice were labeled with a rabbit
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Figure 1 Immunohistochemical findings in mdx skeletal and cardiac muscles. At 1 week after the seventh injection, the 4 mdx mice from
groups 1 and 2 were killed; the quadriceps, diaphragm, gastrocnemius and heart were isolated, blotted dry, trimmed of external tendon,
snap-frozen in liquid N2-cooled isopentane, and stored at 80 1C until further processing. As controls, four untreated mdx mice and four mdx
treated with ZM2-NP alone were also killed. All immunohistochemical procedures were carried out as previously described.11 Dystrophin
appears to be properly expressed at the membrane of muscle fibers of ZM2–AON (M23D)-treated mdx mice. The intensity and localization of
labeling in rescued dystrophin-positive muscle fibers is reminiscent of normal muscle. Dystrophin traces are also visible in mice treated with
the same dose of naked AON. No dystrophin labeling was detected at the sarcolemma in the mdx either untreated or treated only with ZM2NP. Bars indicate the magnifications.
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polyclonal anti-dystrophin antibody (H-300, Santa Cruz
Biotechnology, Santa Cruz, CA, USA) and revealed
with an anti-rabbit fluorescein isothiocyanate-conjugated
secondary antibody (Dako, Glostrup, Denmark). All
samples were double-labeled with a rat monoclonal
antibody for laminin-2 (a2-chain, 4H8-2 Alexis Biochemical, Farmingdale, NY, USA), followed by anti-rat tetramethylrhodamino isothiocyanate-conjugated antibody
(Dako) as internal control.
As counting positive fibers is considered a fundamental parameter for evaluating the efficiency of
dystrophin restoration, we compared two independent
methods to provide a more precise quantification of
dystrophin positivity in muscles. The first method used
was a manual system, the AnalySIS program (Soft
Imaging System, Muenster, Germany),11 which counts
dystrophin-positive fibers with a labeling that covers at
least 50% of the sarcolemma perimeter. The second
method was a novel, semiautomated and semiquantitative analysis system, which calculates the ratio between
dystrophin and laminin a-2-chain-positive areas in
double-labeled samples using novel software developed
in cooperation with Nikon (NIS-Elements 3.0 AR imaging program, Nikon, Firenze, Italy). The mean value of
the dystrophin/laminin a2-chain (fluorescein isothiocyanate/tetramethylrhodamino isothiocyanate) area ratio
was calculated following subtraction of the background.
To provide a graphical representation of the above, mean
values for the fluorescein isothiocyanate/tetramethylrhodamino isothiocyanate area ratio of treated and
untreated mdx mice were normalized to wild-type values
and expressed as a percentage of dystrophin-positive
fibers over the total number of fibers (laminin-a2
positive) (Figure 2a and Supplementary Figure S1).
The two different counting approaches gave quite
similar results. In fact, dystrophin positivity in ZM2–
AON-treated mdx mice was quantified as 18% by manual
count and 16% by the semiautomated system in the
diaphragm, respectively, 26 and 19% in the gastrocnemius, 40 and 35% in the quadriceps and 3 and 6% in the
heart (Figure 2a and Supplementary Figure S1). Dystrophin rescue was found to be significantly improved in
mice treated with ZM2–AON complex with respect to
those treated with naked AON, which showed a
maximum of 11–13% dystrophin-positive fibers in the
quadriceps (Figure 2a and Supplementary Figure S1).
Exon-specific RealTime PCR assays revealed an exon
23-skipping percentage of 4–21% in skeletal muscles and
3% in the heart (Figure 2b): double the % detected by
using the T1-AON compound in skeletal muscles, but
not in the heart.11 Exon 23 skipping was confirmed by
reverse transcription-PCR in all muscles except for the
Figure 2 (a) Immunohistochemical quantification of dystrophin-positive fibers in skeletal muscles (quadriceps, gastrocnemius, diaphragm)
and heart of ZM2–AON- and naked AON-treated mdx mice evaluated by manual counting. Bars represent the percentage of dystrophinpositive fibers in ZM2–AON- (black bars) and naked AON-treated (gray bars) mouse muscles. At least 5000 muscle fibers from each muscle,
obtained from three different levels of tissue blocks for each sample, were studied; data were analyzed using the Mann–Whitney test and the
significance was calculated versus untreated mdx mice. Asterisks indicate statistically significant Po0.05. (b) RealTime reverse transcription
(RT)-PCR analysis (ESRA) for detecting exon 23 skipping. Percentage of exon 23 skipping (y axis) was determined by RealTime PCR in
cardiac and skeletal muscles (quadriceps, gastrocnemius, diaphragm, x axis) from treated mdx animals in comparison with untreated mice.
Histograms represent the percentage of exon 23-skipped transcript (2DDCT values) calibrated on exons 22 and 25, and normalized to wildtype (WT) mice. Black bars: naked AON-treated mice; gray bars: ZM2–AON-treated mice; white bars: mdx mice. (c) Nested RT-PCR analysis
of dystrophin mRNA from the quadriceps, diaphragms, gastrocnemius and hearts of animals in groups 1 and 2 and untreated mice (NT).
Primary and nested PCRs were performed as previously described.11 Full-length dystrophin transcript was detected in all cDNA samples,
and a faint shorter transcript arising from AON-induced exon 23 skipping was observed in all skeletal muscles but not the heart of ZM2–
AON-treated animals and in diaphragm of naked-AON-treated mice. M: molecular Weight Marker IX (Roche, Indianapolis, IN, USA). (d and e)
Dystrophin immunoblot using DYS2 antibody. For WT sample, the total protein loaded was 1/15 of the quantity in the other lanes.
(d) Restored expression of the protein in quadriceps muscle from ZM2–AON- and naked AON-treated mice. Quadriceps from WT and
untreated mdx mice were used as positive and negative controls, respectively. (e) dystrophin protein is clearly visible in the diaphragm from
both naked AON- and ZM2–AON-treated mice and was also detectable, though faintly, in the heart of ZM2–AON-treated mice. WT mouse
diaphragm was used as positive control.
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heart, as expected considering the small amount of skipping
identified by exon-specific RealTime PCR assay (Figure 2c).
Western blotting analysis showed the presence of high
molecular weight dystrophin protein in skeletal muscles
(quadriceps and diaphragm) from ZM2–AON-treated
mdx mice (Figures 2d and e). As expected from the
immunohistochemical and transcriptional data, dystrophin
protein in the heart is visible, albeit poorly, under western
blot (Figure 2e). Interestingly, a lower but detectable
dystrophin restoration was clearly visible under immunohistochemical analysis (Figure 1), although poorly so by
western blotting (Figures 2d and e), at the same dose of
naked AON. Moreover, the total amount (52.5 mg kg1) of
AON injected as ZM2–AON was reduced in comparison with previous studies on naked AON delivery
(120–600 mg kg1).7,8
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Figure 3 Immunolabeling of the smooth muscle arrector pili in treated and untreated mdx mice. The sections of skin biopsies taken from the
region of the scruff of the neck of wild-type (WT), ZM2–AON-treated mdx and untreated mdx mice were labeled with anti-dystrophin
antibody (rabbit polyclonal, Santa Cruz Biotechnology) and incubated with a tetramethylrhodamino isothiocyanate-conjugated secondary
antibody. All samples were double-labeled with an anti-desmin antibody (goat polyclonal 1:10, Santa Cruz Biotechnology) and incubated
with anti-goat fluorescein isothiocyanate-conjugated antibody (1:100, Sigma, St Louis, MO, USA). All samples were observed with a Nikon
Eclipse 80i fluorescence microscope. Dystrophin (red) is clearly visible in the arrector pili smooth muscle of WT mice, absent in untreated
mdx, and rescued in treated mdx. High magnification of treated mdx arrector pili shows an intense and homogeneous labeling of smooth
muscle cells (lower panel). Desmin (green) colocalizes with dystrophin and was expressed in all animals. Bar, 200 mm.
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Regarding PMO–AONs, optimization of naked AON
dose regimens has previously been addressed, and some
dystrophin restoration was detected following intravenous administration of a very low dose of PMO–AONs
(total of 20 mg of PMO per kg).12 Although PMO and
20 OMePS molecules possess different kinetics, and
studies on specific molecules will be required to optimize
dose regimens, our data support the fact that even with
low doses of 20 OMePS, AON dystrophin restoration can
be achieved.
Subsequently, we tested whether dystrophin restoration in treated animals could be detected in the smooth
muscles of the skin arrector pili, as dystrophin expression in human arrector pili smooth muscle has been
previously documented and found to be absent in DMD
patients,13–15 although it had never been studied in the
mdx animal model. We demonstrated that dystrophin is
indeed expressed in the arrector pili smooth muscle of
wild-type mice but absent in the mdx, and that ZM2–
AON-treated mdx mice clearly show protein rescue in
this smooth muscle (Figure 3), unlike mice treated with
an identical dose of naked AON. Furthermore, we
confirmed the expression of dystrophin in human
arrector pili from a healthy subject, as well as its absence
in a DMD patient (Figure 4a). In addition, we explored
whether other sarcolemma proteins, namely a-, b-, d- and
g-sarcoglycans, laminin-a2-chain, dysferlin and caveolin3 were expressed in the arrector pili smooth muscle of
human skin. Accordingly, all skin samples were doublelabeled with anti-desmin antibody (goat polyclonal,
Santa Cruz Biotechnology) and revealed with an antigoat fluorescein isothiocyanate-conjugated antibody. All
sarcoglycans were found to be strongly expressed and
detectable in control human skin arrector pili smooth
muscle (Figure 4b), whereas the other sarcolemma
proteins analyzed (dysferlin, caveolin-3 and laminin-a2chain) were not detected (data not shown). Although
expression of sarcoglycans in smooth muscle has been
well documented, especially in blood vessels,7 their
expression in the smooth muscle of skin arrector pili
has not previously been reported.
The evidence suggests that this novel ZM2–AON
compound greatly enhances dystrophin restoration in
skeletal muscles and slightly enhances it in the cardiac
muscles of mdx mice, thereby confirming that these NPs
represent a very promising vehicle for systemic delivery
Figure 4 Immunolabeling of dystrophin on normal human and
Duchenne muscular dystrophy (DMD) skin smooth muscle arrector
pili. (a) The sections of skin biopsies from a healthy human subject
and the DMD patient were labeled with the anti-dystrophin
antibody (rabbit polyclonal, Santa Cruz Biotechnology) and
incubated with tetramethylrhodamino isothiocyanate (TRITC)-conjugated secondary antibody. To identify smooth muscle cells of
arrector pili, samples were double labeled with an anti-desmin
antibody and revealed with a fluorescein isothiocyanate (FITC)conjugated secondary antibody. Dystrophin was intensely
expressed at the sarcolemma of smooth muscle cells of the arrector
pili of the healthy subject, but absent in the DMD patient. (b)
Immunolabeling of a-, b-, g- and d-sarcoglycans in normal human
skin. Anti-a-, b-, g- and d-sarcoglycan antibodies (1:5, Novocastra,
NewCastle upon Tyne, UK) were revealed with a TRITC-conjugated
antibody. All samples were double-labeled with an anti-desmin
antibody and a FITC-conjugated antibody (1:100, Sigma). Smooth
muscle cells of the arrector pili, identified by desmin labeling, show
an intense labeling with anti-a and -b antibodies. Bar, 20 mm.
Gene Therapy
of AONs. The observed dystrophin rescue was due to a
high skipping percentage (10–20%), remarkable when
one considers the low AON dose delivered, and to the
expression of high molecular weight dystrophin. The
new dystrophin protein was correctly localized at the
Nanoparticle–AON dystrophin rescue
A Ferlini et al
sarcolemma in up to 40% of the fibers, as detected by
immunohistochemical analysis.
The combination of slow release and depot effects,
together with protection from degradation/sequestration
and the high AON-binding capacity of this novel ZM2NP could be responsible for its efficacy and efficiency. A
possible explanation of this depot-release behavior could
be provided by considering the expanded nature of the
shell in water, as AON species form ion pairs with
cationic groups placed within the outer layer or well
inside the shell. Thus, a fast release kinetic could
correspond to the release of the AON species adsorbed
onto the outer shell, whereas the slow desorption process
may be associated to their release from the inner part of
the shell. Hence, the possibility of varying shell chain
density by appropriately adjusting the synthetic parameters of the NP could provide an additional tool for
fine-tuning the depot characteristics of the AON–NP
complex.
Remarkably, we obtained dystrophin restoration with
a very low dose of naked 20 OMePS AON. Optimal dose
regimens of AONs for therapeutic use and their lowest
nontoxic effective dose are still being debated,5,9,11,12 but,
interestingly our results suggest that lower doses of
naked 20 OMePS AONs administered for longer periods
(in our study 7 weeks) might be sufficient to induce low
but detectable dystrophin restoration, similar to that
reported for PMOs, at least in the mdx animal model,
thereby prompting further research in this direction. NPs
are thus confirmed as a very promising delivery system
for AONs, especially due to the fact that, intriguingly, the
skin arrector pili muscle cells were shown to be
susceptible to exon skipping and dystrophin restoration
in ZM2–AON-treated mice. Therefore, if confirmed for
other types of treatment and with different dose regimens, skin smooth muscle could represent a very
promising, less invasive enrichment or even surrogate
end point for verifying dystrophin restoration and
monitoring the efficacy of treatment, not only in animal
models but also in clinical trials.16–19 This would
consequently facilitate approval procedures for the latter
by greatly alleviating the negative impact that compulsory muscle biopsies have on perception of treatment
among patients and their families. In addition, arrector
pili smooth muscle could be considered as a lowinvasive biomarker in other muscular dystrophies such
as sarcoglycanopathies.
In summary, our results show that the ZM2–AON
complex effectively restores dystrophin synthesis in
striated muscles at low doses of AON. Further studies
into the half-life, elimination and administration of NPs
are required to evaluate their eligibility as innovative
nontoxic delivery systems for AON-mediated therapy
in DMD.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The Telethon Italy grants GGP05115, GUP07011 and
GGP09093 (to AF) are acknowledged. Thanks are also
due to Prof A Medici (Department of Chemistry,
University of Ferrara) to the Industria Chimica Emiliana
(ICE Reggio Emilia) Grant (to AF), and to TREAT-NMD
Network of Excellence of EU FP7 n. 036825 (to LM and
Telethon-Italy). We are also grateful to the ISS National
AIDS Program grants, in support of the Nanoparticle
technology platform, awarded to AC, LT and ML. The
ZM2–AON compound and its effects have been patented
at the University of Ferrara, Industrial Liaison Office,
patent IP number TO2009A000782.
437
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Gene Therapy