Gene Therapy (2012) 19, 678–685
& 2012 Macmillan Publishers Limited All rights reserved 0969-7128/12
www.nature.com/gt
REVIEW
Progress in gene therapy of dystrophic heart disease
Y Lai and D Duan
The heart is frequently afflicted in muscular dystrophy. In severe cases, cardiac lesion may directly result in death. Over the
years, pharmacological and/or surgical interventions have been the mainstay to alleviate cardiac symptoms in muscular
dystrophy patients. Although these traditional modalities remain useful, the emerging field of gene therapy has now provided an
unprecedented opportunity to transform our thinking/approach in the treatment of dystrophic heart disease. In fact, the premise
is already in place for genetic correction. Gene mutations have been identified and animal models are available for several types
of muscular dystrophy. Most importantly, innovative strategies have been developed to effectively deliver therapeutic genes to
the heart. Dystrophin-deficient Duchenne cardiomyopathy is associated with Duchenne muscular dystrophy (DMD), the most
common lethal muscular dystrophy. Considering its high incidence, there has been a considerable interest and significant input
in the development of Duchenne cardiomyopathy gene therapy. Using Duchenne cardiomyopathy as an example, here we
illustrate the struggles and successes experienced in the burgeoning field of dystrophic heart disease gene therapy. In light of
abundant and highly promising data with the adeno-associated virus (AAV) vector, we have specially emphasized on AAVmediated gene therapy. Besides DMD, we have also discussed gene therapy for treating cardiac diseases in other muscular
dystrophies such as limb-girdle muscular dystrophy.
Gene Therapy (2012) 19, 678–685; doi:10.1038/gt.2012.10; published online 9 February 2012
Keywords: muscular dystrophy; heart; cardiomyopathy; Duchenne muscular dystrophy; dystrophin; sarcoglycan
INTRODUCTION
Muscular dystrophy refers to a heterogeneous group of inherited
muscle diseases. Patients exhibit progressive muscle wasting and
force decline. Although the molecular pathogenesis of muscular
dystrophy has not been fully elucidated, it is generally agreed that
mechanical injury may play an important role. Contraction-induced
cell shape change distinguishes muscle cells from other body cells.
Deformation creates tremendous stress on the muscle cell membrane.
In the absence of an appropriate protective mechanism, the sarcolemma is broken. Membrane disruption leads to myofiber degeneration, necrosis, fibrosis and eventually muscular dystrophy.
Compared with skeletal muscle, the demand for a stable sarcolemma is even higher for the myocardium because of the constant
pumping activity of the heart. As a result, cardiac involvement is
regularly seen in muscular dystrophy. Cardiomyopathy and/or heart
arrhythmia are typical clinical presentations of dystrophic heart
disease. Lethal consequences include heart failure and sudden cardiac
arrest. Symptom-based therapies have been practiced for years to
attenuate cardiac complications in muscular dystrophy patients. Yet,
palliative treatments offer only limited clinical benefit. The ultimate
cure for dystrophic heart disease will require genetic correction of the
defective genes.
The identification of the disease gene, and development of gene
transfer vector and animal models have established a strong foundation for exploring gene therapy. Genetic mutations in many types of
muscular dystrophy are now defined. Interestingly, a lot of these
mutations involve components of dystrophin-associated glycoprotein
complex (DGC). The DGC is a multiprotein structure that links
cytoskeleton F-actin (g-actin) with laminin in the extracellular matrix.
It mainly consists of dystrophin, a- and b-dystroglycan, and a-, b-,
g- and d-sarcoglycan. Additional components of the DGC include
sarcospan, syntrophin, dystrobrevin and neuronal nitric oxide
synthase.1 Numerous lines of evidence suggest that the DGC may
function as a molecular shock absorber and protect the sarcolemma
from mechanical strain-induced damages.2 Deficiency of the DGC
compromises the cytoskeleton–extracellular matrix connection and
destabilizes the sarcolemma.3,4
An ever-increasing armory of viral and non-viral vectors has been
developed for heart gene transfer.5 Unfortunately, most are either
inefficient or are associated with strong inflammation/immune reaction. The only exception is adeno-associated virus (AAV). AAV is a
non-enveloped single-strand DNA virus.6 Wild-type AAV contains an
B5-kb genome. Except for the B145-bp inverted terminal repeat
(ITR) at the ends, the entire viral genome can be replaced with a
therapeutic expression cassette. Early experimentation with AAV
serotype-2 and -5 showed persistent cardiac transduction following
direct myocardial injection, trans-coronary delivery or intra-cavity
injection.7–9 Now, a broad range of naturally occurring and synthetic
AAV capsids have been engineered for cardiac targeting.5 Impressively,
these newly generated AAV serotypes (such as AAV-6, 8 and 9) result
in robust gene expression in the heart even if delivered through a
peripheral vein.10–14
Establishment of reliable animal models is another crucial factor. It
provides a stage for pre-clinical testing. Several rodent and canine
models have been carefully characterized. They recapitulate representative clinical features of dystrophic heart diseases. Some commonly
used models are dystrophin-null mdx mice and dogs and cadiomyopathic hamsters.15–19
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO, USA
Correspondence: Professor D Duan, Department of Molecular Microbiology and Immunology, The University of Missouri, School of Medicine, One Hospital Dr M610G, MSB,
Columbia, MO 65212, USA.
E-mail: [email protected]
Received 22 November 2011; revised 17 January 2012; accepted 17 January 2012; published online 9 February 2012
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
679
The majority of current gene therapy studies are focused on
dystrophin-deficient cardiomyopathy. Some studies have also been
conducted for d-sarcoglycan-null cardiomyopathy. For this reason, we
have emphasized these topics in this review article. The experience
learned from these studies will provide valuable guidance for gene
therapy of cardiac diseases in other types of muscular dystrophy.
DUCHENNE CARDIOMYOPATHY GENE THERAPY
Duchenne cardiomyopathy refers to cardiac diseases seen in Duchenne
muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and
X-linked dilated cardiomyopathy (XLDC). The common genetic
underpinning is dystrophin deficiency. DMD is the most common
and severe form of muscular dystrophy. In DMD dystrophin is
completely lost in all striated muscles. BMD refers to a mild form
caused by partial loss of dystrophin (quantitative or qualitative).
In XLDC, dystrophin is selectively removed from the heart. Dilated
cardiomyopathy is the characteristic clinical manifestation in Duchenne
cardiomyopathy. Patients may die from heart failure and/or ventricular arrhythmia. As dystrophin gene mutation is the molecular
culprit in Duchenne cardiomyopathy, replacement or repair of the
defective dystrophin gene has been considered as a promising cure.
Gene replacement with synthetic dystrophin genes
Gene delivery to dystrophin-deficient heart. Several AAV serotypes
have been tested in mdx mice, a widely used mouse model. The first
successful gene delivery to the mdx heart was achieved with AAV-5.8
Virus was directly injected into the cardiac chamber in hypothermiashocked neonatal mdx mice. Although meaningful as a proof-ofprinciple, the invasive protocol used in the study is unlikely going to
translate to human patients. Recently, several groups evaluated AAV-6
and AAV-9. Impressive whole heart transduction was obtained following systemic gene delivery. Importantly, these serotypes were not only
effective in neonatal and young adult mdx mice but they also worked
in the heart of aged mdx mice.20–26
Dystrophin gene abbreviation. The full-length dystrophin protein is
encoded in an B14-kb mRNA. It contains four structural domains,
including the N-terminal, rod, cysteine-rich and C-terminal domains.
The rod domain is further divided into 24 spectrin-like repeats and
four intervening hinges. The size of the dystrophin gene presents a
great challenge to AAV-mediated gene transfer.
Much effort has been devoted to the development of minimized
synthetic dystrophin genes.27,28 The goal is to generate abbreviated
dystrophin variants that are small enough for viral vector packaging
but yet retain the critical functional domains. Mini-dystrophin and
micro-dystrophin are two categories currently under investigation.
The minigene is B6–8 kb and it carries X50% of the coding sequence.
In mini-dystrophin, part of the rod domain is removed. The microgene is B4 kb and it contains only about one-third of the coding
sequence. Micro-dystrophin loses a significant portion of the rod
domain and the C-terminal domain. In terms of AAV gene transfer,
the biggest difference is that the microgene can fit into a single AAV
virion but the minigene requires the dual vector system for delivery.29
Gene therapy of Duchenne cardiomyopathy with mini-dystrophin.
Several pairs of dual AAV vectors have been developed to express
mini-dystrophin.23,30–32 In dual vector system, a complete minigene
cassette (including the promoter, minigene and polyadenylation
signal) is engineered into two independent AAV viruses. Mini-dystrophin expression is achieved after co-infection and inter-viral recombination. The first working set of the minigene dual vectors was built
with the trans-splicing strategy.32 In this system, minigene expression
is reconstituted via ITR-mediated end-to-end vector genome joining
and subsequent splicing using pre-engineered splicing signals.33,34
Two independent sets of over-lapping mini-dystrophin vectors were
reported recently.23,30 In the over-lapping approach, the minigene is
split into two overlapping fragments and separately packaged. Reconstitution is achieved through the shared region of homology.35 The
hybrid dual vectors are a new addition.31 It allows reconstitution
through either ITR-mediated end joining (trans-splicing) or transgene sequence-mediated homologous recombination (over-lapping).
Several minigene hybrid vectors have been described.30,34 Currently,
mini-dystrophin dual AAV vectors have only been tested for treating
DMD skeletal muscle disease. Their therapeutic efficacy for Duchenne
cardiomyopathy remains to be determined.
The possibility of using mini-dystrophin to treat Duchenne cardiomyopathy has only been investigated in transgenic mdx mice.36 Cardiac
specific mini-dystrophin expression was achieved in mdx mice using the
alpha-myosin heavy chain promoter.37 Mini-dystrophin completely
prevented sarcolemmal leakage and myocardial fibrosis.36 At 22 months
of age, several ECG parameters (including the PR interval, QT interval
and cardiomyopathy index) were normalized. Cardiac catheter assay
also revealed significant improvement. End-systolic volume, maximal
pressure, dP/dt max and ejection fraction were indistinguishable from
those of normal mice. Nevertheless, dP/dt min, stroke volume and
cardiac output remained suboptimal.36
Gene therapy of Duchenne cardiomyopathy with micro-dystrophin.
Until now, most Duchenne cardiomyopathy gene therapy studies
were conducted with the AAV micro-dystrophin vector.8,21,22,24,26,38
The first evidence supporting microgene therapy in the heart was
provided by Yue et al.8 The authors delivered an AAV-5 microgene
vector to newborn mdx heart. Micro-dystrophin restored DGC
expression and improved sarcolemmal integrity in cardiomyocytes
in vivo.8 Subsequent electrocardiographic study provided evidence of
physiological benefits in neonatal mdx mice.24 A single intravenous
injection of the AAV-9 microgene vector transduced the entire heart in
neonatal mdx mice. The heart rate, PR interval and QT interval were
normalized up to four months after treatment.24
Two studies examined AAV micro-dystrophin therapy in young
adult (4–10-week-old) mdx mice.22,26 One study (Shin et al.) used
AAV-9 and the other (Townsend et al.) used AAV-6. Robust myocardial transduction was observed following systemic gene transfer in
both studies. Shin et al.26 observed a significant improvement of the
heart rate and PR interval for up to 6 months. They also noticed a
reduction of cardiac fibrosis and normalization of fractional shortening on echocardiography.26 ECG was not performed in Townsend
et al. study.22 However, hemodynamic assay at 10 weeks post AAV
therapy showed an increased end-diastolic volume. Further, AAV
microgene treated mice were protected from dobutamine stressinduced acute heart failure.22
One caveat of above studies is that newborn or young adult mdx
mice do not show overt signs of dilated cardiomyopathy.17 Further,
male and female mdx mice exhibit different cardiac phenotypes and
this difference has not been appreciated.16 Characteristic dilated
cardiomyopathy has only been demonstrated in very old female
mdx mice.16,17 A recent study by Bostick et al.21 evaluated AAV
microgene therapy in aged female mdx mice. AAV-9 microgene vector
was delivered to 19-months-old female mdx mice. Histopathology and
cardiac function were examined four months later. Despite ongoing
cardiac disease, impressive AAV transduction was observed throughout the entire heart. Myocardial fibrosis was significantly reduced in
treated mdx mice. Importantly, beneficial ECG and hemodynamic
Gene Therapy
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
680
changes were observed and dobutamine-induced cardiac death was
prevented in all treated mice.21 In summary, evidence so far strongly
supports further development of Duchenne cardiomyopathy gene
replacement therapy with minimized dystrophins.
Repair dystrophin gene through exon skipping
Several thousands of mutations have been reported in DMD
patients.39,40 A common theme of these mutations is that they all
disrupt the open reading frame.41,42 Exon skipping is designed to
modulate splicing with antisense oligonucleotides (AON). Removal of
mutated and/or adjacent exon(s) restores the open reading frame. The
resulting transcript yields an internally truncated, but likely functional
protein. There has been tremendous progress in DMD exon skipping
in last few years.43 Positive results were obtained from several clinical
trials in treating skeletal muscle disease.44–47
A long-standing hurdle of exon-skipping is its inefficiency in the
heart.48 Minimal dystrophin expression was observed in the mdx heart
even after repeated delivery for 1 year.49,50 It is now clear that the
problem is the poor uptake of AON by cardiomyocytes.43 Increasing
AON dose to the extreme level seems to have resulted in some
improvement.51 However, such an approach is clinically unsustainable.52
Another approach to enhance AON delivery is via chemical modification. Using AON that is conjugated to cell penetrating peptides,
non-peptide polymers or nanoparticles, several groups have now
demonstrated widespread myocardial exon skipping in mdx mice.53–62
Interestingly, peptide-conjugated AON did not induce cardiac exon
skipping in dystrophin/utrophin double knockout mice, a much severe
DMD model.63 Future studies are needed to clarify this difference.
Cardiac function has been evaluated in a few exon-skipping studies
that used peptide conjugated AON. Wu et al.53 treated 4-months-old
mdx mice for 3 weeks. Treatment significantly protected mdx mice
from dobutamine-induced cardiac dysfunction and death.53 Jearawiriyapaisarn et al.59 also noticed beneficial echocardiographic changes in
young adult mdx mice. As the heart of young adult mdx mice is only
mildly affected, future studies in a symptomatic model (such as aged
female mdx mice) may provide more rigorous support for exon
skipping mediated Duchenne cardiomyopathy gene therapy. Other
issues that also need the attention are the potential toxicity and
immunogenicity of modified AONs.52,64 Addressing these issues may
pave the way for future clinical translation.
Alternative gene therapy strategies for Duchenne cardiomyopathy
As the primary cause of Duchenne cardiomyopathy is dystrophin
deficiency, it is logical to emphasize on strategies that aim at restoring
dystrophin expression (mini- and micro-dystrophin replacement or
gene repair with exon skipping). However, there are some limitations.
First is the immune response to viral vector and/or newly expressed
dystrophin. Recent studies in dystrophin-deficient dogs and human
DMD patients suggest that untoward immune rejection is a major
obstacle.65–68 Another issue is the incomplete recovery of cardiac
function.36 Little is known about the structure–function relationship
of dystrophin in the heart. Cardiac-specific domain(s) have been
suspected but never been clarified.69,70 In the absence of this knowledge, it is very unlikely that the current gene replacement/repair
strategies will fully meet the need. As a matter of fact, one minidystrophin gene that is known to normalize skeletal muscle force did
not completely restore heart function.36 Exploring alternative gene
therapy strategies may enhance dystrophin-based therapies.
Utrophin for Duchenne cardiomyopathy gene therapy. Utrophin is a
homolog of dystrophin.71 Despite some important differences between
Gene Therapy
two proteins,72,73 many lines of evidence suggest that utrophin may
greatly improve muscle function in dystrophin-deficient subjects.74
Several studies also suggest that utrophin may have helped preserving
heart function in young adult mdx mice and heterozygous mdx
mice.17,75 As utrophin is already expressed in DMD patients, exogenous utrophin expression from a gene therapy vector will likely be
tolerated by our immune system. Similar to dystrophin, the large size
of the utrophin gene also presents a significant challenge to gene
delivery. Development of minimized synthetic utrophin genes may
overcome this hurdle.76,77
Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a). SERCA2a is
an enzyme that pumps cytosolic calcium back to the sarcoplasmic
reticulum in the heart. There are several advantages in developing
SERCA2a-based gene therapy for Duchenne cardiomyopathy. As an
endogenous gene, it posses the same immune advantage as utrophin
(less immunogenic). Unlike utrophin, the SERCA2a gene readily fits
into a single AAV vector.78 Accumulated evidence suggests that
calcium overloading may represent a major pathogenic mechanism
in Duchenne cardiomyopathy.79 Forced expression of SERCA2a via
AAV gene transfer may restore calcium homeostasis and improve
cardiac contractility. Only one study has tested the therapeutic efficacy
of SERCA2a in the mdx heart. Shin et al.79 delivered AAV SERCA2a
vector to 12-m-old mdx mice and examined ECG eight months later.
Significant improvement was observed in treated mice.79
Recent progresses have lent more support to SERCA2a gene
therapy. First, AAV SERCA2a gene therapy has shown encouraging
results in patients with advanced heart failure.78,80,81 Second, it was
found that SERCA2a is highly regulated by a protein called small
ubiquitin-related modifier 1 (SUMO1).82 AAV-mediated SUMO1
overexpression offers another opportunity to elevate SERCA2a expression/activity and hence improve heart function.82
Other possible candidate genes for Duchenne cardiomyopathy gene
therapy. Besides utrophin and SERCA2a, many other endogenous
genes may also be worth considering. Some examples include
AAV-mediated vascular endothelial growth factor expression and
AON-mediated microRNA-208 knockdown.83,84 These strategies
have been shown to treat other types of dilated cardiomyopathy.
Although not tested yet for Duchenne dilated cardiomyopathy,
they may be worth pursuing either alone or in combination with
dystrophin replacement/repair therapy.
Additional considerations in Duchenne cardiomyopathy
gene therapy
Therapeutic threshold. Dystrophin is lost in all cardiomyocytes in
Duchenne cardiomyopathy. However, in a gene therapy scenario we
may not be able to correct every single cell in the heart. It is thus
important to determine the minimal level of gene transfer needed for
the treatment. Heterozygous mdx carriers display a mosaic pattern of
dystrophin expression in approximately 50% of cardiomyocytes.
Interestingly, these mice are completely protected from cardiomyopathy.17,85 At 21 months of age, there are minimal signs of histological
lesions. Further, cardiac function (ECG and hemodynamics) is normalized.17 Human DMD carriers also show 50% mosaic dystrophin
expression. Importantly, the majority does not develop cardiomyopathy.86–89 Taken together, it seems reasonable to set a 50% transduction rate as the therapeutic threshold.
Currently, there is no concrete data on whether expression below
50% can protect the heart. Interestingly, a recent exon skipping study
revealed a quite surprising finding.50 The investigators induced o5%
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
681
dystrophin positive cells in the heart in adult mdx mice. No change
was found in cardiac histology and baseline function. However, an
improved response was noticed under dobutamine stress.50
Therapeutic time window. Therapeutic time window is an important
consideration. Dystrophin expression has been detected during
embryonic development in both rodent and human in both skeletal
muscle and the heart.90–93 One study in mdx mice even suggests that
dystrophin deficiency may severely impair myogenesis.94 However,
this may not necessarily mean the therapeutic window will be missed
if gene therapy is not initiated during fetal development. Several lines
of evidence suggest that embryonic dystrophin deficiency does not
have immediate developmental consequences. For example, neonatal
mdx mice appear normal.95 Similarly, dystrophic symptoms have not
been observed in newborn DMD patients.96 It is possible that
predominant utrophin expression during embryogenesis may have
compensated for the loss of dystrophin.97–100
Transgenic study with the alpha-myosin heavy chain promoter also
suggests that prenatal therapy is not absolutely required for heart
disease amelioration.36 This promoter is not active in the ventricle
during fetal development.37,101 Yet, cardiomyopathy is significantly
mitigated in mini-dystrophin transgenic mice that are under transcriptional regulation of the alpha-myosin heavy chain promoter.36
Taken together, additional studies are needed to determine whether
absence of dystrophin leads to irreversible developmental defects. In
the meantime, considering exciting preclinical data already published,
it is certainly worth to further pursue postnatal gene therapy. On the
other side, with the advance in prenatal and newborn screening,
attempts on fetal and/or neonatal gene transfer should also be
encouraged. Such an approach may allow immediate therapy following early diagnosis.
One interesting concern on fetal and/or neonatal gene transfer is the
loss of the episomal AAV vector genome during postnatal heart
growth. Although a valid concern, it may not be a serious problem.
Cardiomyocyte regeneration is essentially terminated after the first
week of birth.102–104 Postnatal heart size increase is mainly due to
hypertrophy rather than hyperplasia.104
Impact of skeletal muscle disease and smooth muscle dystrophin
deficiency on Duchenne cardiomyopathy gene therapy. Different from
diseases that only affect the heart, there is often concurrent involvement of skeletal muscle in muscular dystrophy-associated heart
diseases. It has been hotly debated whether severity of skeletal muscle
disease influences the outcome of cardiomyopathy and whether
treating skeletal muscle disease benefits or harms the heart.105,106
Several mouse studies have revealed a direct correlation between
skeletal muscle disease and cardiomyopathy. Genetic inactivation of
skeletal muscle-specific myogenic transcription factor myoD not only
worsens skeletal muscle disease, it also induces a severe cardiac
phenotype in young adult mdx mice.106,107 Recently, Crisp et al.108
published a very intriguing result. The authors performed skeletal
muscle specific exon skipping in dystrophin-null mice. Although
dystrophin was not restored in the heart, the authors reported
normalization of heart function by magnetic resonance imaging
assay (unfortunately, no histological data were presented).108 The
authors concluded that there is no need to treat the heart.108 In
other words, cardiac dysfunction will recover automatically when
skeletal muscle disease is under control.
This is quite surprising as a similar exon skipping study by a
different group showed just the opposite result.49 Using histology
examination, Malerba et al.49 found a more severe cardiac pathology
in mdx mice that were only treated for skeletal muscle disease by exon
skipping. Along the same line, Townsend et al.109 demonstrated that
transgenic rescue of skeletal muscle disease alone worsens heart
function in young adult mdx mice. In summary, studies from Malerba
et al. and Townsend et al. suggest that the heart has to be treated.
Curing only skeletal muscle may aggravate heart disease. Clinical data
from XLDC also suggests that we may indeed need to treat the heart.
Dystrophin expression is compromised in the heart but not skeletal
muscle in XLDC.110 Despite a lack of apparent skeletal muscle disease,
XLDC patients develop severe dilated cardiomyopathy.110 Apparently,
additional studies are needed to resolve the conflict. In the mean time,
considering all the pros and cons, the primary focus for Duchenne
cardiomyopathy gene therapy should set to restore dystrophin expression in the heart, or to treat both skeletal muscle and the heart
simultaneously.
Another interesting consideration is the coronary vasculature.
Under normal condition, dystrophin is also expressed in vascular
smooth muscle.91,111,112 Loss of dystrophin induces remarkable biomechanical changes in the vasculature.113–115 Recent studies further
suggest that selective restoration of dystrophin in smooth muscle or
increasing the vasculature alone may reduce DMD skeletal muscle
disease.116,117 However, therapeutic effect of dystrophin expression in
the coronary vasculature has not been determined. As systemic AAV
gene delivery has been shown to transduce smooth muscle,10 it is
possible that the success of current AAV micro-dystrophin gene
therapy may partially attribute to the restoration of dystrophin in
the coronary vasculature.21,22,24,26 Nevertheless, direct experimental
evidence is lacking. Future studies are needed to either support or
reject this intriguing hypothesis.
GENE THERAPY OF CARDIOMYOPATHY IN LIMB-GIRDLE
MUSCULAR DYSTROPHY 2F (LGMD2F)
LGMD2F is an extremely rare form of muscular dystrophy.118 It is
caused by d-sarcoglycan deficiency. Although most patients show
skeletal muscle dysfunction and late-onset cardiomyopathy, a subgroup of patients develop severe dilated cardiomyopathy without
obvious skeletal muscle illness. Despite the scarce incidence, d-sarcoglycan deficient cardiomyopathy has been frequently used in proof-ofprinciple gene therapy studies. The reason for this is two-fold. The
animal models are readily available and the d-sarcoglycan gene is fairly
small. Naturally occurring cardiomyopathic hamsters have been
around for several decades.119 These hamsters carry a deletion mutation in the d-sarcoglycan gene. They exhibit hypertrophic (BIO 14.6
strain) or dilated (TO-2 strain) cardiomyopathy.18 In addition to the
hamster model, two independent lines of d-sarcoglycan knockout
mice have also been generated.120,121 The small size of the gene is
another advantage. It allows easy packaging into any viral vector
including AAV.
Early studies used adenovirus to express the d-sarcoglycan
gene.122,123 Efficient myocardial transduction is achieved but expression does not last long.123 This obstacle is overcome with AAV vectors.
Persistent d-sarcoglycan expression was first obtained in the hearts of
TO-2 and BIO14.6 hamsters using AAV-2. A drawback of AAV-2 is
that it requires highly invasive technique (such as intramyocardial
injection and ex vivo coronary perfusion) for gene delivery.124–126
Systemic gene delivery is clinically more appealing. This strategy was
initially tested with an AAV-8 d-sarcoglycan vector.127 Efficient myocardial transduction was observed following intraperiotoneal and
intravenous injection in neonatal and young adult TO-2 hamsters.
Histopathology (myocardial fibrosis, calcification, and necrosis) was
completely eliminated and heart function was normalized on echocardiography examination.127 Recently, studies from several independent
Gene Therapy
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
682
groups have further confirmed this exciting result in BIO14.6 hamsters
and d-sarcoglycan-null mice with AAV-1, 8 and 9.128–130
Besides d-sarcoglycan gene replacement, investigators have also
explored other alternative gene therapy strategies. In one study,
Hoshijima et al. tested a phospholamban inhibiting AAV-2 vector.
Disturbance of calcium homeostasis has an important role in the
pathogenesis of LGMD2F.118 Phospholamban ablation has been
shown to improve sarcoplasmic reticulum calcium cycling in mouse
cardiomyopathy models.131 Encouragingly, trans-coronary AAV-2
gene therapy enhanced heart function in young adult BIO14.6
hamsters.9 Thus, aiming at downstream events may open another
promising avenue to treat d-sarcoglycan-deficient cardiomyopathy.
Similar to DMD, d-sarcoglycan deficiency also compromises skeletal muscle. Two studies tested whether gene transfer to the heart alone
is sufficient for treating cardiomyopathy in LGMD2F. Goehringer
et al.129 achieved heart-specific expression with a cardiac muscle
specific promoter. Yang et al.132 generated a relatively myocardiumspecific AAV capsid using DNA shuffling technique. Interestingly,
heart restricted expression significantly reduced myocardial fibrosis
and improved cardiac function in d-sarcoglycan-null mice and TO-2
hamsters.129,132
Untoward immune response to newly expressed dystrophin is a
serious concern in DMD gene therapy.65–68 However, immune rejection
has not been encountered in AAV or even adenovirus-mediated
d-sarcoglycan gene delivery in rodent models.122,124,125,127–130 Whether
this will hold up in human patients remains to be seen. Interestingly, a
recent AAV a-sarcoglycan clinical trial has revealed a quite safe immune
profile. No T cell response to a-sarcoglycan was detected.133,134 Nevertheless, caution should be taken when we proceed to human trial.
Collectively, animal study results suggest that gene therapy may
represent a viable approach to treat cardiomyopathy in LGMD2F.
GENE THERAPY OF CARDIAC COMPLICATIONS IN OTHER
TYPES OF MUSCULAR DYSTROPHY
Cardiac complications are universally observed in muscular dystrophies.135,136 However, besides dystrophin-deficient cardiomyopathy
and d-sarcoglycan-deficient cardiomyopathy, gene therapy has been
barely explored to treat heart diseases in other types of muscular
dystrophy. With the promising pre-clinical results in Duchenne and
LGMD2F cardiomyopathy, it is expected that there will be a growing
interest in testing gene therapy in other dystrophic heart diseases.
Several points should be considered in the design of these studies. First
is the nature of the mutation. Many muscular dystrophies are caused
by dominant mutations. In these cases, the strategy should be focused
on the elimination of the abnormal allele (for example, via RNA
interference) rather than gene supplementation.137,138 Another issue is
the clinical presentation. In Emery–Dreifuss muscular dystrophy and
myotonic dystrophy, arrhythmia rather than dilated cardiomyopathy
is more frequently encountered.136 Innovative means are needed to
tailor gene therapy protocols to meet different clinical situations.
Third, despite the lack of heart-focused gene therapy studies, there
have been considerable progresses in gene therapy of skeletal muscle
diseases in many muscular dystrophies. The lessons learned from these
studies should be incorporated in the development of dystrophic heart
disease gene therapy.
SUMMARY AND PERSPECTIVE
With the ability to reverse the fundamental genetic defect, gene
therapy has demonstrated immense potential to cure dystrophic
heart disease. A strong foundation has been established from treating
cardiac complications in rodent models of Duchenne cardiomyopathy
Gene Therapy
and LGMD2F. With the ongoing research activities, we have no doubt
that more powerful gene delivery vectors (such as an AAV vector with
reduced immunogenicity/improved transduction efficiency) will
emerge in the coming years. However, despite the rosy picture, there
remain significant obstacles for an unequivocal clinical success in the
near future. For LGMD2F, because of the extreme low incidence it will
be a great challenge to assemble enough patients for a standard clinical
trial. For DMD, reproducing murine results in dystrophic dogs may
represent a more realistic goal.28
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The research in the authors’ laboratory was supported by grants from the
National Institutes of Health HL-91883 (DD), Muscular Dystrophy Association
(DD), Parent Project Muscular Dystrophy (DD) and Jesse’s Journey: The
Foundation for Gene and Cell Therapy (DD).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Ehmsen J, Poon E, Davies K. The dystrophin-associated protein complex. J Cell Sci
2002; 115: 2801–2803.
Ervasti JM. Dystrophin, its interactions with other proteins, and implications for
muscular dystrophy. Biochim Biophys Acta 2007; 1772: 108–117.
Dalkilic I, Kunkel LM. Muscular dystrophies: genes to pathogenesis. Curr Opin Genet
Dev 2003; 13: 231–238.
Heydemann A, McNally EM. Consequences of disrupting the dystrophin-sarcoglycan
complex in cardiac and skeletal myopathy. Trends Cardiovasc Med 2007; 17: 55–59.
Wasala NB, Shin JH, Duan D. The evolution of heart gene delivery vectors. J Gene Med
2011; 13: 557–565.
Carter BJ. Adeno-associated virus and the development of adeno-associated virus
vectors: a historical perspective. Mol Ther 2004; 10: 981–989.
Svensson EC, Marshall DJ, Woodard K, Lin H, Jiang F, Chu L et al. Efficient and
stable transduction of cardiomyocytes after intramyocardial injection or intracoronary
perfusion with recombinant adeno-associated virus vectors. Circulation 1999; 99:
201–205.
Yue Y, Li Z, Harper SQ, Davisson RL, Chamberlain JS, Duan D. Microdystrophin gene
therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves
sarcolemma integrity in the Mdx mouse heart. Circulation 2003; 108: 1626–1632.
Hoshijima M, Ikeda Y, Iwanaga Y, Minamisawa S, Date MO, Gu Y et al. Chronic
suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat Med 2002; 8: 864–871.
Bostick B, Ghosh A, Yue Y, Long C, Duan D. Systemic AAV-9 transduction in mice is
influenced by animal age but not by the route of administration. Gene Therapy 2007;
14: 1605–1609.
Inagaki K, Fuess S, Storm TA, Gibson GA, McTiernan CF, Kay MA et al. Robust
systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer
superior to that of AAV8. Mol Ther 2006; 14: 45–53.
Pacak CA, Mah CS, Thattaliyath BD, Conlon TJ, Lewis MA, Cloutier DE et al.
Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo. Circ Res 2006; 99: e3–e9.
Wang Z, Zhu T, Qiao C, Zhou L, Wang B, Zhang J et al. Adeno-associated virus serotype
8 efficiently delivers genes to muscle and heart. Nat Biotechnol 2005; 23: 321–328.
Gregorevic P, Blankinship MJ, Allen JM, Crawford RW, Meuse L, Miller DG et al.
Systemic delivery of genes to striated muscles using adeno-associated viral vectors.
Nat Med 2004; 10: 828–834.
Fine DM, Shin JH, Yue Y, Volkmann D, Leach SB, Smith BF et al. Age-matched
comparison reveals early electrocardiography and echocardiography changes in dystrophin-deficient dogs. Neuromuscul Disord 2011; 21: 453–461.
Bostick B, Yue Y, Duan D. Gender influences cardiac function in the mdx model of
Duchenne cardiomyopathy. Muscle Nerve 2010; 42: 600–603.
Bostick B, Yue Y, Long C, Duan D. Prevention of dystrophin-deficient cardiomyopathy
in twenty-one-month-old carrier mice by mosaic dystrophin expression or complementary dystrophin/utrophin expression. Circ Res 2008; 102: 121–130.
Sakamoto A, Ono K, Abe M, Jasmin G, Eki T, Murakami Y et al. Both hypertrophic and
dilated cardiomyopathies are caused by mutation of the same gene, delta-sarcoglycan,
in hamster: an animal model of disrupted dystrophin-associated glycoprotein complex.
Proc Natl Acad Sci USA 1997; 94: 13873–13878.
Smith BF, Yue Y, Woods PR, Kornegay JN, Shin JH, Williams RR et al. An intronic
LINE-1 element insertion in the dystrophin gene aborts dystrophin expression and
results in Duchenne-like muscular dystrophy in the corgi breed. Lab Invest 2011; 91:
216–231.
Gregorevic P, Blankinship MJ, Allen JM, Chamberlain JS. Systemic microdystrophin
gene delivery improves skeletal muscle structure and function in old dystrophic mdx
mice. Mol Ther 2008; 16: 657–664.
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
683
21 Bostick B, Shin J-H, Yue Y, Duan D. AAV-microdystrophin therapy improves cardiac
performance in aged female mdx mice. Mol Ther 2011; 19: 1826–1832.
22 Townsend D, Blankinship MJ, Allen JM, Gregorevic P, Chamberlain JS, Metzger JM.
Systemic administration of micro-dystrophin restores cardiac geometry and prevents
dobutamine-induced cardiac pump failure. Mol Ther 2007; 15: 1086–1092.
23 Odom GL, Gregorevic P, Allen JM, Chamberlain JS. Gene Therapy of mdx mice with
large truncated dystrophins generated by recombination using rAAV6. Mol Ther 2011;
19: 36–45.
24 Bostick B, Yue Y, Lai Y, Long C, Li D, Duan D. Adeno-associated virus serotype-9
microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx
mice. Hum Gene Ther 2008; 19: 851–856.
25 Ghosh A, Yue Y, Shin J-H, Duan D. Systemic trans-splicing AAV delivery efficiently
transduces the heart of adult mdx mouse, a model for Duchenne muscular dystrophy.
Hum Gene Ther 2009; 20: 1319–1328.
26 Shin JH, Nitahara-Kasahara Y, Hayashita-Kinoh H, Ohshima-Hosoyama S, Kinoshita
K, Chiyo T et al. Improvement of cardiac fibrosis in dystrophic mice by rAAV9mediated microdystrophin transduction. Gene Therapy 2011; 18: 910–919.
27 Chamberlain JS. Gene therapy of muscular dystrophy. Hum Mol Genet 2002; 11:
2355–2362.
28 Duan D. Duchenne muscular dystrophy gene therapy: lost in translation? Res Rep Biol
2011; 2: 31–42.
29 Duan D. From the smallest virus to the biggest gene: marching towards gene therapy
for Duchenne muscular dystrophy. Discov Med 2006; 6: 103–108.
30 Zhang Y, Duan D. Novel mini-dystrophin gene dual adeno-associated virus vectors
restore neuronal nitric oxide synthase expression at the sarcolemma. Hum Gene Ther
2012; 23: 98–103.
31 Ghosh A, Yue Y, Lai Y, Duan D. A hybrid vector system expands aden-associated viral
vector packaging capacity in a transgene independent manner. Mol Ther 2008; 16:
124–130.
32 Lai Y, Yue Y, Liu M, Ghosh A, Engelhardt JF, Chamberlain JS et al. Efficient in vivo
gene expression by trans-splicing adeno-associated viral vectors. Nat Biotechnol
2005; 23: 1435–1439.
33 Duan D, Yan Z, Engelhardt JF. Expanding the capacity of AAV vectors. In: Bloom ME,
Cotmore SF, Linden RM, Parrish CR, Kerr JR (eds). Parvoviruses. Hodder Arnold;
Distributed in the USA by Oxford University Press: London, New York, 2006,
pp 525–532.
34 Ghosh A, Duan D. Expending adeno-associated viral vector capacity: a tale of two
vectors. Biotechnol Genet Eng Rev 2007; 24: 165–177.
35 Duan D, Yue Y, Engelhardt JF. Expanding AAV packaging capacity with trans-splicing
or overlapping vectors: a quantitative comparison. Mol Ther 2001; 4: 383–391.
36 Bostick B, Yue Y, Long C, Marschalk N, Fine DM, Chen J et al. Cardiac expression of a
mini-dystrophin that normalizes skeletal muscle force only partially restores heart
function in aged Mdx mice. Mol Ther 2009; 17: 253–261.
37 Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific
regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol
Chem 1991; 266: 24613–24620.
38 Gregorevic P, Allen JM, Minami E, Blankinship MJ, Haraguchi M, Meuse L et al.
rAAV6-microdystrophin preserves muscle function and extends lifespan in severely
dystrophic mice. Nat Med 2006; 12: 787–789.
39 Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT.
Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of
mutation types and paradoxical cases that confirm the reading-frame rule. Muscle
Nerve 2006; 34: 135–144.
40 Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard
MT et al. Mutational spectrum of DMD mutations in dystrophinopathy patients:
application of modern diagnostic techniques to a large cohort. Hum Mutat 2009;
30: 1657–1666.
41 Koenig M, Beggs AH, Moyer M, Scherpf S, Heindrich K, Bettecken T et al. The
molecular basis for Duchenne versus Becker muscular dystrophy: correlation of
severity with type of deletion. Am J Hum Genet 1989; 45: 498–506.
42 Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM. An explanation for
the phenotypic differences between patients bearing partial deletions of the DMD
locus. Genomics 1988; 2: 90–95.
43 Lu QL, Yokota T, Takeda S, Garcia L, Muntoni F, Partridge T. The status of Exon
skipping as a therapeutic approach to Duchenne muscular dystrophy. Mol Ther 2011;
19: 9–15.
44 Goemans NM, Tulinius M, van den Akker JT, Burm BE, Ekhart PF, Heuvelmans N et al.
Systemic administration of PRO051 in Duchenne’s muscular dystrophy. N Engl J Med
2011; 364: 1513–1522.
45 van Deutekom JC, Janson AA, Ginjaar IB, Frankhuizen WS, Aartsma-Rus A, BremmerBout M et al. Local dystrophin restoration with antisense oligonucleotide PRO051.
N Engl J Med 2007; 357: 2677–2686.
46 Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, Anthony K et al. Exon
skipping and dystrophin restoration in patients with Duchenne muscular dystrophy
after systemic phosphorodiamidate morpholino oligomer treatment: an open-label,
phase 2, dose-escalation study. Lancet 2011; 378: 595–605.
47 Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C et al. Local
restoration of dystrophin expression with the morpholino oligomer AVI-4658 in
Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation,
proof-of-concept study. Lancet Neurol 2009; 8: 918–928.
48 Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD et al. Systemic delivery of
morpholino oligonucleotide restores dystrophin expression bodywide and improves
dystrophic pathology. Nat Med 2006; 12: 175–177.
49 Malerba A, Boldrin L, Dickson G. Long-term systemic administration of unconjugated
morpholino oligomers for therapeutic expression of dystrophin by exon skipping in
skeletal muscle: implications for cardiac muscle integrity. Nucleic Acid Ther 2011;
21: 293–298.
50 Wu B, Xiao B, Cloer C, Shaban M, Sali A, Lu P et al. One-year treatment of morpholino
antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx
mice. Mol Ther 2011; 19: 576–583.
51 Wu B, Lu P, Benrashid E, Malik S, Ashar J, Doran TJ et al. Dose-dependent restoration
of dystrophin expression in cardiac muscle of dystrophic mice by systemically
delivered morpholino. Gene Therapy 2010; 17: 132–140.
52 Goyenvalle A, Davies KE. Challenges to oligonucleotides-based therapeutics for
Duchenne muscular dystrophy. Skelet Muscle 2011; 1: 8.
53 Wu B, Moulton HM, Iversen PL, Jiang J, Li J, Spurney CF et al. Effective rescue of
dystrophin improves cardiac function in dystrophin-deficient mice by a modified
morpholino oligomer. Proc Natl Acad Sci USA 2008; 105: 14814–14819.
54 Yin H, Lu Q, Wood M. Effective exon skipping and restoration of dystrophin expression
by peptide nucleic acid antisense oligonucleotides in mdx mice. Mol Ther 2008; 16:
38–45.
55 Yin H, Moulton HM, Seow Y, Boyd C, Boutilier J, Iverson P et al. Cell-penetrating
peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac
dystrophin expression and function. Hum Mol Genet 2008; 17: 3909–3918.
56 Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S et al.
Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers
in the muscles of mdx mice. Mol Ther 2008; 16: 1624–1629.
57 Wu B, Li Y, Morcos PA, Doran TJ, Lu P, Lu QL. Octa-guanidine Morpholino Restores
Dystrophin Expression in Cardiac and Skeletal Muscles and Ameliorates Pathology in
Dystrophic mdx Mice. Mol Ther 2009; 17: 864–871.
58 Ferlini A, Sabatelli P, Fabris M, Bassi E, Falzarano S, Vattemi G et al. Dystrophin
restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2
NP-AON complexes. Gene Therapy 2010; 17: 432–438.
59 Jearawiriyapaisarn N, Moulton HM, Sazani P, Kole R, Willis MS. Long-term improvement in mdx cardiomyopathy after therapy with peptide-conjugated morpholino
oligomers. Cardiovasc Res 2010; 85: 444–453.
60 Yin H, Moulton HM, Betts C, Seow Y, Boutilier J, Iverson PL et al. A fusion peptide
directs enhanced systemic dystrophin exon skipping and functional restoration in
dystrophin-deficient mdx mice. Hum Mol Genet 2009; 18: 4405–4414.
61 Yin H, Moulton HM, Betts C, Merritt T, Seow Y, Ashraf S et al. Functional rescue of
dystrophin-deficient mdx mice by a chimeric peptide-PMO. Mol Ther 2010; 18:
1822–1829.
62 Yin H, Saleh AF, Betts C, Camelliti P, Seow Y, Ashraf S et al. Pip5 transduction
peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in
heart and phenotypic correction in mdx mice. Mol Ther 2011; 19: 1295–1303.
63 Goyenvalle A, Babbs A, Powell D, Kole R, Fletcher S, Wilton SD et al. Prevention of
dystrophic pathology in severely affected dystrophin/utrophin-deficient mice by morpholino-oligomer-mediated exon-skipping. Mol Ther 2010; 18: 198–205.
64 Amantana A, Moulton HM, Cate ML, Reddy MT, Whitehead T, Hassinger JN et al.
Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptidemorpholino oligomer conjugate. Bioconjug Chem 2007; 18: 1325–1331.
65 Shin JH, Yue Y, Srivastava A, Smith B, Lai Y, Duan D. A simplified immune
suppression scheme leads to persistent micro-dystrophin expression in Duchenne
muscular dystrophy dogs. Hum Gene Ther 2012; 23: e-pub ahead of print 14
December 2011; doi:10.1089/hum.2011.147.
66 Wang Z, Allen JM, Riddell SR, Gregorevic P, Storb R, Tapscott SJ et al. Immunity to
adeno-associated virus-mediated gene transfer in a random-bred canine model of
Duchenne muscular dystrophy. Hum Gene Ther 2007; 18: 18–26.
67 Wang Z, Kuhr CS, Allen JM, Blankinship M, Gregorevic P, Chamberlain JS et al.
Sustained AAV-mediated dystrophin expression in a canine model of Duchenne
muscular dystrophy with a brief course of immunosuppression. Mol Ther 2007; 15:
1160–1166.
68 Mendell JR, Campbell K, Rodino-Klapac L, Sahenk Z, Shilling C, Lewis S et al.
Dystrophin immunity in Duchenne’s muscular dystrophy. N Engl J Med 2010; 363:
1429–1437.
69 Kaspar RW, Allen HD, Ray WC, Alvarez CE, Kissel JT, Pestronk A et al. Analysis of
dystrophin deletion mutations predicts age of cardiomyopathy onset in becker
muscular dystrophy. Circ Cardiovasc Genet 2009; 2: 544–551.
70 Jefferies JL, Eidem BW, Belmont JW, Craigen WJ, Ware SM, Fernbach SD et al.
Genetic predictors and remodeling of dilated cardiomyopathy in muscular dystrophy.
Circulation 2005; 112: 2799–2804.
71 Blake DJ, Weir A, Newey SE, Davies KE. Function and genetics of dystrophin and
dystrophin-related proteins in muscle. Physiol Rev 2002; 82: 291–329.
72 Li D, Bareja A, Judge L, Yue Y, Lai Y, Fairclough R et al. Sarcolemmal nNOS anchoring
reveals a qualitative difference between dystrophin and utrophin. J Cell Sci 2010;
123: 2008–2013.
73 Rybakova IN, Humston JL, Sonnemann KJ, Ervasti JM. Dystrophin and utrophin bind
actin through distinct modes of contact. J Biol Chem 2006; 281: 9996–10001.
74 Hirst RC, McCullagh KJ, Davies KE. Utrophin upregulation in Duchenne muscular
dystrophy. Acta Myol 2005; 24: 209–216.
75 Janssen PM, Hiranandani N, Mays TA, Rafael-Fortney JA. Utrophin deficiency worsens
cardiac contractile dysfunction present in dystrophin-deficient mdx mice. Am J
Physiol Heart Circ Physiol 2005; 289: H2373–H2378.
76 Odom GL, Gregorevic P, Allen JM, Finn E, Chamberlain JS. Microutrophin delivery
through rAAV6 increases lifespan and improves muscle function in dystrophic
dystrophin/utrophin-deficient mice. Mol Ther 2008; 16: 1539–1545.
Gene Therapy
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
684
77 Tinsley JM, Potter AC, Phelps SR, Fisher R, Trickett JI, Davies KE. Amelioration of the
dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature
1996; 384: 349–353.
78 Hajjar RJ, Zsebo K, Deckelbaum L, Thompson C, Rudy J, Yaroshinsky A et al. Design
of a phase 1/2 trial of intracoronary administration of AAV1/SERCA2a in patients with
heart failure. J Card Fail 2008; 14: 355–367.
79 Shin JH, Bostick B, Yue Y, Hajjar R, Duan D. SERCA2a gene transfer improves
electrocardiographic performance in aged mdx mice. J Transl Med 2011; 9: 132.
80 Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B et al. Calcium
upregulation by percutaneous administration of gene therapy in cardiac
disease (CUPID Trial), a first-in-human phase 1/2 clinical trial. J Card Fail 2009;
15: 171–181.
81 Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B et al. Calcium
upregulation by percutaneous administration of gene therapy in cardiac
Disease (CUPID): a Phase 2 Trial of Intracoronary Gene Therapy of Sarcoplasmic
Reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation 2011;
124: 304–313.
82 Kho C, Lee A, Jeong D, Oh JG, Chaanine AH, Kizana E et al. SUMO1-dependent
modulation of SERCA2a in heart failure. Nature 2011; 477: 601–605.
83 Pepe M, Mamdani M, Zentilin L, Csiszar A, Qanud K, Zacchigna S et al. Intramyocardial VEGF-B167 gene delivery delays the progression towards congestive
failure in dogs with pacing-induced dilated cardiomyopathy. Circ Res 2010; 106:
1893–1903.
84 Montgomery RL, Hullinger TG, Semus HM, Dickinson BA, Seto AG, Lynch JM et al.
Therapeutic inhibition of miR-208a improves cardiac function and survival during
heart failure. Circulation 2011; 124: 1537–1547.
85 Yue Y, Skimming JW, Liu M, Strawn T, Duan D. Full-length dystrophin expression in
half of the heart cells ameliorates beta-isoproterenol-induced cardiomyopathy in mdx
mice. Hum Mol Genet 2004; 13: 1669–1675.
86 Hoogerwaard EM, Bakker E, Ippel PF, Oosterwijk JC, Majoor-Krakauer DF, Leschot NJ
et al. Signs and symptoms of Duchenne muscular dystrophy and Becker muscular
dystrophy among carriers in The Netherlands: a cohort study. Lancet 1999; 353:
2116–2119.
87 Grain L, Cortina-Borja M, Forfar C, Hilton-Jones D, Hopkin J, Burch M. Cardiac
abnormalities and skeletal muscle weakness in carriers of Duchenne and Becker
muscular dystrophies and controls. Neuromuscul Disord 2001; 11: 186–191.
88 Nolan MA, Jones OD, Pedersen RL, Johnston HM. Cardiac assessment in childhood
carriers of Duchenne and Becker muscular dystrophies. Neuromuscul Disord 2003;
13: 129–132.
89 Holloway SM, Wilcox DE, Wilcox A, Dean JC, Berg JN, Goudie DR et al. Life
expectancy and death from cardiomyopathy amongst carriers of Duchenne and Becker
muscular dystrophy in Scotland. Heart 2008; 94: 633–636.
90 Houzelstein D, Lyons GE, Chamberlain J, Buckingham ME. Localization of dystrophin
gene transcripts during mouse embryogenesis. J Cell Biol 1992; 119: 811–821.
91 Hoffman EP, Hudecki MS, Rosenberg PA, Pollina CM, Kunkel LM. Cell and fiber-type
distribution of dystrophin. Neuron 1988; 1: 411–420.
92 Ginjaar IB, Viragh S, Markman MW, van Ommen GJ, Moorman AF. Dystrophin
expression in the developing conduction system of the human heart. Microsc Res
Tech 1995; 30: 458–468.
93 Wessels A, Ginjaar IB, Moorman AF, van Ommen GJ. Different localization of
dystrophin in developing and adult human skeletal muscle. Muscle Nerve 1991;
14: 1–7.
94 Merrick D, Stadler LK, Larner D, Smith J. Muscular dystrophy begins early in
embryonic development deriving from stem cell loss and disrupted skeletal muscle
formation. Dis Model Mech 2009; 2: 374–388.
95 Gillis JM. Understanding dystrophinopathies: an inventory of the structural and
functional consequences of the absence of dystrophin in muscles of the mdx
mouse. J Muscle Res Cell Motil 1999; 20: 605–625.
96 Emery AEH, Muntoni F. Duchenne Muscular Dystrophy, 3rd edn. Oxford University
Press: Oxford, New York, 2003, x, 270pp.
97 Love DR, Byth BC, Tinsley JM, Blake DJ, Davies KE. Dystrophin and dystrophinrelated proteins: a review of protein and RNA studies. Neuromuscul Disord 1993; 3:
5–21.
98 Schofield J, Houzelstein D, Davies K, Buckingham M, Edwards YH. Expression of the
dystrophin-related protein (utrophin) gene during mouse embryogenesis. Dev Dyn
1993; 198: 254–264.
99 Rigoletto C, Prelle A, Ciscato P, Moggio M, Comi G, Fortunato F et al. Utrophin
expression during human fetal development. Int J Dev Neurosci 1995; 13: 585–593.
100 Mora M, Di Blasi C, Barresi R, Morandi L, Brambati B, Jarre L et al. Developmental
expression of dystrophin, dystrophin-associated glycoproteins and other membrane
cytoskeletal proteins in human skeletal and heart muscle. Brain Res Dev Brain Res
1996; 91: 70–82.
101 Ng WA, Grupp IL, Subramaniam A, Robbins J. Cardiac myosin heavy chain
mRNA expression and myocardial function in the mouse heart. Circ Res 1991;
68: 1742–1750.
102 Steinhauser ML, Lee RT. Regeneration of the heart. EMBO Mol Med 2011; 3:
701–712.
103 Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN et al.
Transient regenerative potential of the neonatal mouse heart. Science 2011; 331:
1078–1080.
104 Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from
hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;
28: 1737–1746.
Gene Therapy
105 Shin J-H, Bostick B, Yue Y, Duan D. Duchenne cardiomyopathy gene therapy. In: Duan
D (ed). Muscle Gene Therapy. Springer Science+Business Media, LLC: New York,
2010, pp 141–162.
106 Duan D. Challenges and opportunities in dystrophin-deficient cardiomyopathy gene
therapy. Hum Mol Genet 2006; 15 (Spec No 2): R253–R261.
107 Megeney LA, Kablar B, Perry RL, Ying C, May L, Rudnicki MA. Severe cardiomyopathy
in mice lacking dystrophin and MyoD. Proc Natl Acad Sci USA 1999; 96: 220–225.
108 Crisp A, Yin H, Goyenvalle A, Betts C, Moulton HM, Seow Y et al. Diaphragm rescue
alone prevents heart dysfunction in dystrophic mice. Hum Mol Genet 2011; 20:
413–421.
109 Townsend D, Yasuda S, Li S, Chamberlain JS, Metzger JM. Emergent dilated
cardiomyopathy caused by targeted repair of dystrophic skeletal muscle. Mol Ther
2008; 16: 832–835.
110 Cohen N, Muntoni F. Multiple pathogenetic mechanisms in X linked dilated cardiomyopathy. Heart 2004; 90: 835–841.
111 Nguyen TM, Ellis JM, Love DR, Davies KE, Gatter KC, Dickson G et al. Localization of
the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of
dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating
brain cell lines. J Cell Biol 1991; 115: 1695–1700.
112 Rivier F, Robert A, Hugon G, Mornet D. Different utrophin and dystrophin properties
related to their vascular smooth muscle distributions. FEBS Lett 1997; 408: 94–98.
113 Dye WW, Gleason RL, Wilson E, Humphrey JD. Altered biomechanical properties of
carotid arteries in two mouse models of muscular dystrophy. J Appl Physiol 2007;
103: 664–672.
114 Loufrani L, Levy BI, Henrion D. Defect in microvascular adaptation to chronic changes
in blood flow in mice lacking the gene encoding for dystrophin. Circ Res 2002; 91:
1183–1189.
115 Loufrani L, Matrougui K, Gorny D, Duriez M, Blanc I, Levy BI et al. Flow (shear stress)induced endothelium-dependent dilation is altered in mice lacking the gene encoding
for dystrophin. Circulation 2001; 103: 864–870.
116 Ito K, Kimura S, Ozasa S, Matsukura M, Ikezawa M, Yoshioka K et al. Smooth musclespecific dystrophin expression improves aberrant vasoregulation in mdx mice. Hum
Mol Genet 2006; 15: 2266–2275.
117 Verma M, Asakura Y, Hirai H, Watanabe S, Tastad C, Fong GH et al. Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased
vasculature in mdx mice. Hum Mol Genet 2010; 19: 4145–4159.
118 Blain AM, Straub VW. delta-Sarcoglycan-deficient muscular dystrophy: from discovery
to therapeutic approaches. Skelet Muscle 2011; 1: 13.
119 Gertz EW. Cardiomyopathic Syrian hamster: a possible model of human disease. Prog
Exp Tumor Res 1972; 16: 242–260.
120 Coral-Vazquez R, Cohn RD, Moore SA, Hill JA, Weiss RM, Davisson RL et al.
Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle:
a novel mechanism for cardiomyopathy and muscular dystrophy. Cell 1999; 98:
465–474.
121 Hack AA, Lam MY, Cordier L, Shoturma DI, Ly CT, Hadhazy MA et al. Differential
requirement for individual sarcoglycans and dystrophin in the assembly and function
of the dystrophin-glycoprotein complex. J Cell Sci 2000; 113: 2535–2544.
122 Holt KH, Lim LE, Straub V, Venzke DP, Duclos F, Anderson RD et al. Functional rescue
of the sarcoglycan complex in the BIO 14.6 hamster using delta-sarcoglycan gene
transfer. Mol Cell 1998; 1: 841–848.
123 Ikeda Y, Gu Y, Iwanaga Y, Hoshijima M, Oh SS, Giordano FJ et al. Restoration of
deficient membrane proteins in the cardiomyopathic hamster by in vivo cardiac gene
transfer. Circulation 2002; 105: 502–508.
124 Li J, Wang D, Qian S, Chen Z, Zhu T, Xiao X. Efficient and long-term intracardiac gene
transfer in delta-sarcoglycan-deficiency hamster by adeno-associated virus-2 vectors.
Gene Therapy 2003; 10: 1807–1813.
125 Toyo-oka T, Kawada T, Xi H, Nakazawa M, Masui F, Hemmi C et al. Gene therapy
prevents disruption of dystrophin-related proteins in a model of hereditary dilated
cardiomyopathy in hamsters. Heart Lung Circ 2002; 11: 174–181.
126 Kawada T, Nakazawa M, Nakauchi S, Yamazaki K, Shimamoto R, Urabe M et al.
Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene
therapy: amelioration of morphological findings, sarcolemmal permeability, cardiac
performances, and the prognosis of TO- 2 hamsters. Proc Natl Acad Sci USA 2002;
99: 901–906.
127 Zhu T, Zhou L, Mori S, Wang Z, McTiernan CF, Qiao C et al. Sustained whole-body
functional rescue in congestive heart failure and muscular dystrophy hamsters by
systemic gene transfer. Circulation 2005; 112: 2650–2659.
128 Vitiello C, Faraso S, Sorrentino NC, Di Salvo G, Nusco E, Nigro G et al. Disease rescue
and increased lifespan in a model of cardiomyopathy and muscular dystrophy by
combined AAV treatments. PLoS One 2009; 4: e5051.
129 Goehringer C, Rutschow D, Bauer R, Schinkel S, Weichenhan D, Bekeredjian R et al.
Prevention of cardiomyopathy in delta-sarcoglycan knockout mice after systemic
transfer of targeted adeno-associated viral vectors. Cardiovasc Res 2009; 82:
404–410.
130 Hoshijima M, Hayashi T, Jeon YE, Fu Z, Gu Y, Dalton ND et al. Delta-sarcoglycan gene
therapy halts progression of cardiac dysfunction, improves respiratory failure, and
prolongs life in myopathic hamsters. Circ Heart Fail 2011; 4: 89–97.
131 Chu G, Kranias EG. Phospholamban as a therapeutic modality in heart failure.
Novartis Found Symp 2006; 274: 156–171; discussion 172–175, 272–276.
132 Yang L, Jiang J, Drouin LM, Agbandje-McKenna M, Chen C, Qiao C et al.
A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and
in vivo selection. Proc Natl Acad Sci USA 2009; 106: 3946–3951.
Targeting dystrophic cardiomyopathy
Y Lai and D Duan
685
133 Mendell JR, Rodino-Klapac LR, Rosales XQ, Coley BD, Galloway G, Lewis S et al.
Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle
muscular dystrophy, type 2D. Ann Neurol 2010; 68: 629–638.
134 Mendell JR, Rodino-Klapac LR, Rosales-Quintero X, Kota J, Coley BD, Galloway G
et al. Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan
and associated proteins. Ann Neurol 2009; 66: 290–297.
135 Hermans MC, Pinto YM, Merkies IS, de Die-Smulders CE, Crijns HJ, Faber CG. Hereditary
muscular dystrophies and the heart. Neuromuscul Disord 2010; 20: 479–492.
136 Beynon RP, Ray SG. Cardiac involvement in muscular dystrophies. QJM 2008; 101:
337–344.
137 Wallace LM, Garwick SE, Harper SQ. RNAi therapy for dominant muscular dystrophies
and other myopathies. In: Duan D (ed). Muscle Gene Therapy. Springer Science+Business Media, LLC: New York, 2010, pp 99–115.
138 Wallace LM, Garwick-Coppens SE, Tupler R, Harper SQ. RNA interference improves
myopathic phenotypes in mice over-expressing FSHD Region Gene 1 (FRG1). Mol
Ther 2011; 19: 2048–2054.
Gene Therapy