Review Article Antisense oligonucleotides for the treatment of cardiomyopathy in Duchenne muscular dystrophy
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Am J Transl Res 2019;11(3):1202-1218
www.ajtr.org /ISSN:1943-8141/AJTR0085739
Review Article
Antisense oligonucleotides for the treatment of
cardiomyopathy in Duchenne muscular dystrophy
Quynh Nguyen1, Toshifumi Yokota1,2
1
Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, 8812-112 St.,
Edmonton, AB T6G 2H7, Canada; 2The Friends of Garret Cumming Research and Muscular Dystrophy Canada HM
Toupin Neurological Science Research Chair, 8812-112 St., Edmonton, AB T6G 2H7, Canada
Received September 19, 2018; Accepted January 2, 2019; Epub March 15, 2019; Published March 30, 2019
Abstract: Duchenne muscular dystrophy (DMD) is an X-linked recessive fatal neuromuscular disorder characterized
by progressive muscle degeneration which affects one in 3500-5000 males born worldwide. DMD is caused by
loss-of-function mutations in the dystrophin (DMD) gene encoding for dystrophin, a cytoskeletal protein that sup-
ports the structural integrity of myofibers during cycles of muscle contraction and relaxation. DMD patients do not
only experience skeletal muscle deterioration but also severe cardiomyopathy, which is recognized as the current
leading cause of death for the disease. Among the therapies being developed, exon skipping using antisense oligo-
nucleotides (AOs) is one of the most promising approaches. AOs effectively restore dystrophin expression in skeletal
muscles; however, they are highly inefficient in the heart due to endosomal entrapment. Improving skeletal muscle
function without restoring dystrophin expression in cardiac tissue may exacerbate cardiomyopathy due to increased
voluntary activity. This review consolidates the preclinical antisense approaches to improve dystrophin restoration,
with a special focus on the heart.
Keywords: Duchenne muscular dystrophy, dystrophin, cardiomyopathy, antisense therapy, morpholinos, drug de-
livery
Introduction damage, patients with DMD have markedly ele-
vated serum levels of creatine kinase (CK), even
Duchenne muscular dystrophy (DMD) is an at birth [11]. Death usually occurs in their 20s
X-linked recessive disorder characterized by due to respiratory or cardiac complications
progressive muscle degeneration [1]. The prev- [11-14].
alence of DMD is estimated to be 1 in 3500-
5000 live male births, making it the most com- DMD is caused by mutations in the DMD gene,
mon lethal neuromuscular disorder [2, 3]. Pa- which encodes a membrane-associated protein
tients with DMD generally stay asymptomatic at called dystrophin. Dystrophin has 4 domains:
birth although they could show signs of delayed an actin-binding N-terminal domain, a rod do-
gross motor development compared to their main consisting of 24 spectrin-like repeat
peers [4-6]. The disease progresses rapidly, motifs for structural flexibility, a cysteine-rich
with muscle weakness and wasting observed domain for facilitating protein-protein interac-
first in the proximal muscles then extending to tion, and a C-terminal domain for binding sarco-
the more distal muscles [7]. Affected boys lemmal proteins [15, 16]. As a member of the
often lose ambulation by the age of 12 and dystrophin-glycoprotein complex (DGC), dystro-
experience multiple organ system dysfunction phin functions to link the actin cytoskeleton of
such as neuromuscular scoliosis, joint contrac- muscle cells to the extracellular matrix, provid-
ture, osteoporosis, restrictive lung disease, ing mechanical support and membrane stabili-
obstructive sleep apnea, cardiomyopathy, and zation during muscle contraction and relaxation
psychological problems [8-10]. The age of cycles [15, 17-20]. In the absence of dystrophin,
onset and the rate of decline are highly variable muscle fibers experience mechanical stress
among patients. Due to continuous muscle and become susceptible to tearing and frag-Antisense therapy for DMD cardiomyopathy
mentation, resulting in degeneration [21, 22]. has been pushed into the late 20s [12, 13].
Other studies have suggested that dystrophin Since respiratory dysfunction is now better
also serves non-mechanical roles [23]. managed, cardiomyopathy is presently the lea-
ding cause of death among DMD patients.
The DMD gene is located on the short arm of Cardiomyopathy is estimated to present in 25%
the X chromosome (Xp21.3-p21.2) spanning of patients at the age of 6, and 59% of patients
2.4 Mb with 79 exons which produces a 14 kb at the age of 10 [27, 46]. The majority of
transcript [24-26]. The DMD gene has 3 main patients will have developed cardiomyopathy
promoters that produce full-length dystrophin by 18 years of age. Recognition of signs and
(M, B, and P), with other promoters responsible symptoms of cardiac dysfunction can be chal-
for the transcription of different, smaller iso- lenging since DMD patients are usually wheel-
forms [27, 28]. The M promoter produces the chair-bound and do not perform increased car-
Dp427m isoform which is expressed in cardiac diac workload [47]. The correlation between a
and skeletal muscle, the B promoter produces patient’s genotype and the severity of their car-
the Dp427c isoform which is expressed in neu- diac phenotype remains uncertain [48-52].
rons of the cortex and the Cornu Ammonis (CA)
region of the hippocampus, and the P promoter The earliest clinical signs of cardiomyopathy
produces the Dp427p isoform which is ex- are either decreased systolic function or sinus
pressed in the Purkinje cells of the brain. Other tachycardia, with the latter commonly seen in
shorter isoforms include Dp260 expressed in teenage DMD patients [53, 54]. Sinus tachycar-
the retina, Dp140 expressed in the kidney and dia results in elevated heart rate, which in-
brain, Dp116 expressed in the peripheral creases the workload of the already deteriorat-
nerves and Dp71 which is ubiquitously ex- ing dystrophic myocardium [55-57]. Arrhyth-
pressed [29-33]. mias are a common cardiac involvement in
patients with DMD and are significantly corre-
DMD is considered one of the largest genes in
lated with decreased heart function. App-
the human genome. Furthermore, it is located
roximately 44% of DMD patients show signs of
in a genomic region with high rates of recombi-
arrhythmias [58]. Supraventricular tachycardia
nation [34-36]. Due to its length and location,
(SVT) and ventricular tachycardia (VT) are
DMD is highly susceptible to mutation. Ap-
observed in 10% of individuals with DMD [58].
proximately 60% of DMD cases are due to dele-
Although DMD cardiomyopathy is routinely
tions of one or more exons, ~6% due to duplica-
tions, and the rest due to small insertions/dele- described as dilated cardiomyopathy (DCM),
tions or point mutations [11, 16, 37, 38]. These patients with decreased systolic or diastolic
mutations usually disrupt the reading frame or function do not necessarily show ventricular
introduce a premature stop codon, both of dilation [49, 59, 60]. Additionally, myocardial
which lead to the absence of dystrophin. There fibrosis usually precedes left ventricular dys-
is no significant correlation between mutation function (LVD) [61, 62]. The age of onset and
size and disease severity [39-41]. Most muta- the rate of progression are variable for LVD.
tions that maintain the DMD reading frame pro- However, patients with onset of LVD before 18
duce truncated yet partly functional dystrophin, years of age were found to have a significantly
and often result in a milder form of the disease shorter lifespan compared to those who had
known as Becker muscular dystrophy (BMD) LVD after 18 years old [48]. Other features of
[42-44]. Although the skeletal muscle symp- DMD cardiomyopathy include abnormal cardiac
toms are less severe, the majority of BMD conduction (as caused by, for example, vacuole
patients also develop cardiomyopathy, and it is degeneration in the Purkinje fibers), congestive
the leading cause of death in patients with heart failure and sudden cardiac arrest [27, 28,
BMD [45]. 47, 63-66].
Cardiomyopathy in DMD patients Myocardial issues in DMD patients are unavoid-
able since dystrophin serves the same func-
The life expectancy for patients with DMD used tions in cardiomyocytes as in skeletal muscle
to be less than 20 years of age just two decades cells [67]. In the absence of dystrophin, cardio-
ago. Due to advancements in ventilator support myocytes experience increased structural vul-
and spinal surgery, the average age of mortality nerability and are susceptible to membra-
1203 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
Figure 1. Antisense-induced exon skipping in DMD. Deletion of exon 49 and 50 in the DMD gene creates a frame-
shift mutation which results in a premature stop codon and no dystrophin produced. In the case shown, AOs can
specifically bind to exon 51 and interfere with the splicing machinery to exclude exon 51 from the mature mRNA,
thereby restoring the reading frame and producing truncated but partly functional dystrophin.
ne instability and tearing [22]. Cardiomyocyte other interventions such as ventricular assist
remodeling occurs secondary to myocardial devices (VADs) or cardiac transplantation [9,
wasting, which leads to ventricular enlarge- 47, 82-92]. Treatment with corticosteroids has
ment and fibrosis [68]. As previously men- been shown to improve cardiac function. DMD
tioned, affected hearts do not always show patients who received steroids such as pred-
signs of ventricular dilation. Another conse- nisolone/prednisone or deflazacort had signifi-
quence of myocardial tearing and fragmenta- cantly delayed onset of cardiomyopathy, less
tion is the dysregulation of ion influx/efflux in ventricular dilation and systolic dysfunction
the heart [69, 70]. Notably, damages to the than those who did not. However, patients
myocardial membrane disrupt Ca2+ homeosta- using corticosteroids can experience some
sis. Since Ca2+ acts as a second messenger in long-term complications such as weight gain,
many cellular pathways, Ca2+ dysregulation re- bone fractures, cataracts, etc. [9, 10, 93-96],
sults in multiple downstream abnormalities which limits their use for therapy. ACEIs and
within myocardial tissue. One significant conse- ARBs have proven effective in reducing left ven-
quence of Ca2+ dysregulation includes increa- tricular hypertrophy and fibrosis. Furthermore,
sed reactive oxygen species (ROS) production initiation of beta-adrenergic receptor blocker
and mitochondrial dysfunction. Elevated Ca2+ treatment along with ACEIs has been demon-
levels within cardiomyocytes activate the mito- strated to improve left ventricular systolic func-
chondria to produce ROS which leads to apop- tion in DMD patients [88, 91, 97]. However,
tosis and further muscle wasting [69-79]. An most of these studies have their inherent limi-
important second messenger affected by Ca2+ tations, which makes it challenging for data
dysregulation is nitric oxide (NO) which has interpretation as well as treatment application
roles in blood flow and vasoregulation [80, 81]. in clinical settings.
Increased intracellular Ca2+ concentrations
also enhance the risk of Ca-dependent arrhyth- Antisense oligonucleotides for the treatment
mias and cellular Ca2+ overload in DMD patients of DMD
[69].
At present, there is no cure for DMD. Current
Cardiomyopathy in DMD patients is currently treatments are mostly palliative, aiming at alle-
not curative. Therapies commonly used include: viating the symptoms of the disease [11, 14].
corticosteroids, angiotensin-converting enzyme Several promising approaches have been in-
inhibitors (ACEIs), angiotensin II receptor block- vestigated such as utrophin up-regulation, viral
ers (ARBs), beta-adrenergic receptor blockers, gene therapy, cell-based therapy, antisense oli-
mineralocorticoid receptor antagonists and gonucleotides (AOs) for exon skipping and,
1204 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
Figure 2. Chemical structures commonly used in antisense therapy. (A) RNA, (B) 2’-OMe-phosphorothioate, (C) Tricy-
clo-DNA, (D) Phosphorodiamidate morpholino oligomers. X or Y can be a peptide; Y can be an octaguanidine moiety
(E) as seen in Vivo-morpholino.
most recently, CRISPR-Cas9-mediated gene sistance to nuclease degradation, but also
editing [69]. In this review, we will discuss the enhance their pharmacological properties. Al-
use of AOs in the treatment of DMD with a spe- terations at the 2’ position of the ribose sugar
cific focus on treating the heart. have yielded a class of AOs with improved safe-
ty and efficacy profiles. 2’-O-methyl (2’-OMe)
AOs are short, synthetic nucleic acid sequenc- and 2’-O-methoxyethyl (2’-MOE) alkyl substitu-
es which bind to complementary target mRNA ents are the most studied members of this
sequences and lead to either endonuclease- group. In addition, the use of a phosphorothio-
mediated transcript knockdown or splice mod- ate backbone which contains a sulfur in a non-
ulation [98, 99]. AO-mediated exon skipping bridging location on the phosphate backbone
can correct the reading frame by removing an significantly improves AO nuclease resistance
out-of-frame exon or exons from the DMD pre- and binding affinity to serum proteins [98, 107-
mRNA, producing a truncated but partly func- 109]. Antisense chemistries that contain both
tional dystrophin protein [100-102] (Figure 1). a phosphorothioate backbone and a 2’-alkyl
The first generation of AOs has the unmodified substituent, such as 2’-OMe-phosphorothioate
phosphoribose backbone, making them highly (2’OMePS) AOs, have shown several favorable
susceptible to degradation by nucleases [103, properties compared to their unmodified coun-
104] (Figure 2). Due to their short half-life, first terparts [105, 110-113]. The phosphodiester
generation AOs rarely achieve sufficient intra- backbone can also be replaced with a polyam-
cellular concentrations to have a therapeutic ide structure made up of repeating N-(2-
effect [105, 106]. Second and third generation aminoethyl) glycine units, which has given rise
AOs contain chemically modified structures. to another class of AOs termed peptide nucleic
These modifications not only increase AO re- acids (PNAs) [114]. This new class of AO can
1205 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
sterically block splicing factors, inhibit ribo- and multi-vesicular bodies (MVBs), and lyso-
some recruitment and bind non-coding RNAs, somes. Most AOs are initially delivered to early
thereby further expanding therapeutic potential endosomes. Subsequently, they can be traf-
[69]. ficked to lysosomes for degradation or seques-
tered in late endosomes/MVBs. Antisense dru-
Among these next-generation chemistries, ph- gs trapped within endomembrane compart-
osphorodiamidate morpholino oligomers (PM- ments are pharmacologically inactive as they
Os) represent the most advanced use of anti- remain separated from their target sites by
sense therapy for DMD. PMOs have the deoxy- membrane barriers [106, 129]. Nevertheless,
ribose/ribose moiety replaced by a morpholine a small portion of AOs escapes endosomes and
ring, and the charged phosphodiester inter- only then can they reach their targets and
subunit linkage replaced by an uncharged become pharmacologically active. The concept
phosphorodiamidate linkage, making PMOs of endosomal escape has been widely accept-
nuclease-resistant and charge-neutral [115, ed as the most important barrier to the effec-
116]. One of the challenges with using nucleic tive use of antisense drugs [127, 130]. Im-
acid-based molecules as therapeutics is their portantly for developing heart-effective AOs,
stability and potential toxicity in cells. The the research found that AOs (particularly PMOs)
advantage of being charge-neutral, as in the tend to be trapped in endosomes in cardiomyo-
case of PMOs, is that this imparts an even cytes more than in skeletal muscle cells [131].
greater resistance to nucleases, which typically Since cardiomyopathy is the leading cause of
target charged molecules. Additionally, due to death in DMD patients, researchers have
the lack of charge, PMOs are safer since they looked into different approaches to enhance
are unlikely to activate Toll-like receptors, a delivery of AOs to cardiac tissues. The next sec-
class of receptors involved in producing innate tion will discuss some of the most promising
immune responses against pathogenic materi- approaches to improving AO efficacy for treat-
al [117]. In fact, a PMO targeting DMD exon 51, ing the heart.
called eteplirsen or Exondys 51 (Sarepta
Therapeutics, Cambridge, MA, USA), was condi- Strategies to improve the efficacy of antisense
tionally approved by the U.S. Food and Drug drugs for the treatment of cardiomyopathy in
Administration (FDA) in 2016, and several DMD patients
PMOs targeting other DMD exons, including
golodirsen or SRP-4053 (Sarepta Therapeutics) Tricyclo-DNA
and NS-065/NCNP-01 (NS Pharma, Paramus,
NJ, USA) are currently undergoing clinical trials In the late nineties, a novel class of AOs known
[118-121]. as tricyclo-DNAs (tcDNAs) was shown to display
unique pharmacological properties. tcDNA is a
Although AOs effectively rescue dystrophin conformationally-constrained DNA analog that
expression in skeletal muscles, they are highly deviates from the natural DNA structure by the
inefficient in cardiac muscles [122, 123]. presence of an ethylene bridge connecting C3’
Failure to restore dystrophin expression in the and C5’ [132-134]. tcDNA shows enhanced
heart in the presence of rescued dystrophin in RNA affinity and nuclease resistance, as well
skeletal muscles can exacerbate cardiac issues as the inability to elicit RNase H activity [135-
due to increased patient voluntary activity 138]. Furthermore, tcDNAs can spontaneously
[124, 125]. The major barrier to effective use of form defined nanoparticles which potentia-
AOs in therapy is the delivery of antisense drugs lly improves delivery and cellular uptake com-
to their intracellular targets. Upon reaching the pared to other antisense chemistries [139-
cell surface, AOs are internalized by endocyto- 141].
sis. A variety of cell-surface receptors have
been suggested to bind AOs and facilitate their In a study by Goyenvalle and colleagues, a
entry into the cell including integrins, scavenger mouse model carrying a nonsense mutation in
receptors and Toll-like receptors [126-128]. exon 23 of the Dmd gene (hereafter referred to
Regardless of the endocytosis route taken, AOs as mdx mice) was injected intravenously with a
entering a cell will encounter an intricate net- 15 nucleotide long (15-mer) tcDNA-AO that tar-
work of membrane compartments that include gets the skipping of Dmd exon 23 [139].
early and recycled endosomes, late endosomes Treatment with 200 mg/kg/week of the drug
1206 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
for 12 weeks rescued up to 40% of wild type experiments are mentioned in this paper, but a
(WT) dystrophin expression in the heart. selected few will be discussed for each
Additionally, tcDNA-treated mice showed signif- approach.
icantly higher levels of dystrophin rescue, espe-
cially in the diaphragm and the heart, com- The (RXR)4 peptide (with R standing for arginine
pared to those treated with PMO and 2’-OMe and X standing for aminohexanoic acid) was the
AOs at an equimolar dosage. Similar results first arginine-rich CPP tested in the mdx model
were observed in a dystrophin/utrophin double of DMD [146]. In a study by Yin and colleagues,
knockout mouse model where treatment with (RXR)4 peptides were conjugated to PMOs tar-
weekly intravenous (IV) injections of 200 mg/ geting Dmd exon 23. A single IV injection at 25
kg of tcDNA over a period of 5-20 weeks mg/kg via tail vein resulted in ~50% exon 23
restored 53% of WT dystrophin levels in the skipping in the heart of treated mice. A wide-
heart. Treatment with tcDNA also ameliorated spread, uniform distribution of dystrophin-posi-
cardiac systolic function as demonstrated by tive fibers was also observed in cardiac tissue
improved ventricular ejection fractions and as shown by immunostaining. Western blot
shortening fractions in echocardiography. analysis demonstrated dystrophin restoration
at levels between 10 and 20% of that found in
One of the advantages of tcDNA is its increased the WT mouse heart. Even at lower doses
RNA binding affinity. Due to this feature, shorter administered (weekly injections at 6 mg/kg for
tcDNAs can be designed, reducing the mass of 3 weeks), dystrophin restoration was still
AOs administered and potential AO-induced observed in cardiac tissue as shown by immu-
toxicity while retaining their therapeutic effect. nostaining and Western blotting.
To investigate the efficacy of shorter tcDNAs,
mdx mice were injected intravenously with a Subsequently, another arginine-rich peptide,
13-mer tcDNA targeting Dmd exon 23 at 200 named the B-peptide, demonstrated high lev-
mg/kg/week for 12 weeks. The 13-mer tcDNA els of exon skipping in various muscles includ-
induced particularly high levels of exon skip- ing the heart. The B-peptide contains arginine,
ping and restored dystrophin in the heart as aminohexanoic acid and beta alanine (abbrevi-
shown by RT-PCR, Western blotting and immu- ated B) in an (RXRRBR)2 sequence [147-149]. A
nostaining. The safety profile of the treatment single IV injection at 30 mg/kg of B-peptide-
also showed that tcDNAs were well-tolerated, conjugated PMO (PPMO) restored 94% of dys-
with only minimal renal toxicity and liver inflam- trophin expression in cardiac muscle fibers of
mation observed [138]. Although tcDNAs have treated mdx mice by immunohistochemistry,
the potential to rescue dystrophin in cardiac tis- 50% of WT dystrophin levels by Western blot,
sue, treatment with these AOs requires high and 63% exon 23 skipping by RT-PCR. Repeated
dosage and multiple dose administrations. treatment for 3 months at the same dose at
biweekly intervals further improved dystrophin
Peptides restoration in cardiac muscle. Skipped tran-
script levels increased to 72%. Immunohis-
Among the various delivery systems studied, tochemistry and Western blot demonstrated
cell-penetrating peptides (CPPs) have gained dystrophin rescue at levels comparable to that
the most considerable interest. CPPs are com- in WT hearts. Dystrophin restoration was also
posed of cationic and/or amphipathic amino observed in the muscles of the atria and large
acids that are capable of delivering a wide vessels such as the aorta or vena cava and the
range of nucleic acid-based molecules both in pulmonary arteries. PPMO treatment signifi-
vitro and in vivo [142-144]. These peptides cantly improved cardiac function and prevent-
either possess natural translocating properties ed cardiac failure under dobutamine stress
or were engineered by combining different pro- [150]. A PPMO cocktail was also shown to
tein domains to have these properties [145]. restore dystrophin expression in the myocardi-
Many studies have investigated peptide-conju- um and cardiac Purkinje fibers, and improve
gated AOs for the potential treatment of DMD. cardiac conduction abnormalities in a canine
This review will discuss some of the most prom- model of DMD [151].
ising approaches, namely: arginine-rich pep-
tides, B peptides, PNA/PMO internalization Another promising class of CPPs is the Pips.
peptides (Pips), and phage peptides. Not all Pips are a novel series of transduction peptides
1207 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
developed from mutagenesis and functional also evaluated and no significant changes were
studies of a derivative of Penetratin, with the detected for liver and kidney damage. This
six arginine residues added to the N terminus result is encouraging since the phage peptides
of the CPP (R6Pen) [152]. Penetratin is origi- could be used as an alternative to arginine-rich
nally derived from the homeobox peptide of peptides, which have shown toxicity in higher
Drosophila Antennapedia. The CPPs in the Pip animals [156-158].
series are characterized by a central hydropho-
bic motif anchored on each side by arginine- Octaguanidine morpholino
rich sequences similar to that of the B-peptide
which contains arginine, aminohexanoic acid, In an effort to improve the efficacy of AO sys-
and beta-alanine residues. Among the highly temic delivery and to reduce the immunogenic-
efficient members of this series are Pip5 and ity of cell penetrating peptides, dendrimeric
Pip6, both of which have demonstrated high octaguanidine moieties have been conjugated
levels of dystrophin restoration in various mus- to PMOs to form a new class of AOs named
cles including the heart [153, 154]. A single IV Vivo-Morpholinos or vPMOs [150, 159].
injection of 25 mg/kg of Pip5e-PMO (a member
The guanidine groups in vPMOs work similarly
of the Pip5 family) induced almost complete
to the arginine-rich CPPs found conjugated to
exon skipping in cardiac tissue where full length
PMOs. As such, vPMOs have been shown to
uncorrected transcript was barely detectable. induce high levels of dystrophin expression es-
Immunohistochemistry data revealed more pecially in the heart [149]. Additionally, the
than 90% of dystrophin-positive fibers in cardi- combination of a synthetic scaffold and multi-
ac muscle. Dystrophin restoration was further ple unnatural side chains makes vPMOs unlike-
confirmed by Western blot which revealed more ly to elicit an immune response, thus allowing
than 50% of WT dystrophin levels in the heart for multiple administrations to maintain ade-
[153]. Modifications to the central hydrophobic quate levels of dystrophin in the body [160].
core of the Pip5 peptide gave rise to a novel
derivative of the Pip series, consecutively vPMOs have been shown to rescue cardiac dys-
named Pip6. When directly compared to Pip5e- trophin expression in vivo [161]. A single IV
PMO treatment, a member of the Pip6 family injection of a vPMO targeting mouse Dmd exon
exhibited significantly higher dystrophin resto- 23 (Vivo-ME23) at 6 mg/kg resulted in a strong
ration in the heart of treated mice at very low dystrophin signal in a significant number of car-
doses (12.5 mg/kg). The Pip6 series have also diomyocytes in mdx mice. Repeated treatment
been demonstrated to prevent exercise- at the same dose for five times biweekly res-
induced cardiomyopathy following Pip-PMO cued dystrophin expression in more than 40%
treatment in dystrophic mice [154]. of cardiomyocytes as observed by immunos-
taining. Western blot showed dystrophin rescue
While the above-mentioned CPPs work well for to approximately 10% of normal levels in cardi-
neutral AOs like PMOs, it is challenging to con- ac muscles. vPMOs have also been shown to
jugate positively charged peptides to negative- restore dystrophin expression in a canine mo-
ly-charged AOs such as 2’OMePS due to the del of DMD, however, no systemic injection was
presence of strong electrostatic interactions. In performed so whether vPMOs can rescue dys-
an effort to find uncharged CPPs, Jirka and trophin in canine cardiac tissues remains to be
colleagues screened a phage display peptide determined [162]. Regardless, these encourag-
library and identified a 7-mer peptide (P4) that ing results represent an important step forward
significantly enhanced AO uptake in cardiac tis- in our effort to overcome the challenges associ-
sue [155]. Dystrophic mdx mice were injected ated with the delivery of AOs to the heart.
subcutaneously with 200 mg/kg/week doses
of 2’OMePS-P4 conjugate targeting mouse Ultrasound and microbubbles
Dmd exon 23 for 6 weeks. Treatment with pep-
tide-conjugated AOs resulted in enhanced exon Another promising approach for improving AO
skipping efficacy with a significant difference in delivery to cardiac tissue is the use of ultra-
cardiac tissue compared to naked AOs, possi- sound in combination with contrast-enhancing
bly due to improved uptake in cardiac muscle. microbubbles [163]. Microbubbles are used to
The safety profile of the peptide conjugate was carry the drugs to an area of interest, then
1208 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
ultrasound is applied to burst the microbub- ZM2, have been shown to restore dystrophin
bles, resulting in tissue-specific delivery of the expression in the heart of mdx mice [172, 173].
therapeutic materials [164-167]. Ultrasound
exposure improves the efficacy of intracellular T1 nanoparticles are a type of cationic core-
delivery due to a phenomenon known as sono- shell nanospheres, made up of a core of poly-
poration or cellular sonification. This effect cre- methylmethacrylate (PMMA) and surrounded
ates transient pores in the cellular plasma by a shell of cationic groups, which facilitates
membrane by a process known as inertial cavi- the binding of charged AOs. In a study by
tation, thus allowing bioactive materials to Rimessi and colleagues, T1 nanoparticles were
enter the cells. Inertial cavitation is enhanced demonstrated to deliver 2’OMePS AOs and
by using microbubbles of contrast agents [164, induce dystrophin rescue in body-wide striated
muscles. AOs targeting the boundary sequenc-
167-170]. AOs can be mixed with or incorporat-
es of exon and intron 23 of mouse Dmd (M23D)
ed into microbubbles in a number of ways such
were conjugated with T1 nanoparticles, and
as binding to the microbubble shell or using
delivered by weekly intraperitoneal (IP) injec-
site-specific ligands. The combination of ultra-
tions at 2.7 mg/kg to mdx mice [172]. T1
sound and microbubbles not only improves
nanoparticles showed a wide distribution in the
intracellular delivery but also reduces the
body, including the heart. At one week post-
amount of drugs used systemically thereby le-
injection, immunohistochemical analysis of
ssening the potential toxicity and side effects treated mice showed the presence of dystro-
of treatment. phin-positive cardiomyocytes in various areas
of the heart. Dystrophin was however undetect-
In order to investigate the microbubble proper-
able in cardiac tissues 6 weeks after the last
ties that are important for influencing drug
injection. Total and skipped dystrophin tran-
delivery efficacy in vivo, PMOs targeting Dmd
script levels in the hearts of treated mice were
exon 23 of the mdx mouse were injected sys-
also significantly increased with 80% and 16%
temically at 16 mg/kg via the tail vein [171].
more transcripts, respectively. Although dystro-
PMOs were incorporated into three different
phin was undetected in the heart by Western
commercially available microbubbles: Sona-
blot, the results were positive considering a sig-
zoid, Optison, and SonoVue. Ultrasound was nificantly reduced dose compared to previous
then applied to the heart for two minutes after studies. From a safety aspect, T1 nanoparticles
injections. Treated hearts were collected 24 may cause adverse effects due to their slow
hours or one-week post injection. Of the three biodegradability, indicating a possibility of
microbubbles tested, Sonazoid and Optison accumulative toxicity for long-term treatments
induced significant amounts of dystrophin-pos- [172]. Also, T1 nanoparticles can form small
itive fibers in cardiac tissues. Treatments with aggregates and are therefore not suitable for IV
PMOs only (no ultrasound or microbubbles) injections.
were ineffective in terms of restoring dystro-
phin in cardiomyocytes. Microbubbles were To improve the efficacy of T1 nanoparticles, a
also found to increase AO delivery to the heart more efficient class of nanoparticles termed
in a dose-dependent manner. Histological anal- ZM2 was designed [173]. ZM2 nanoparticles
ysis showed no signs of tissue damage or are composed of a PMMA core and a random
observable morphological differences between copolymer shell consisting of units derived
samples treated with ultrasound and microbub- from N-isopropylacrylamide+ (NIPAM) and reac-
bles and non-treated controls. Additionally, tive methacrylate-bearing cationic groups. mdx
microbubble stability was determined to be the mice were injected IP weekly for 7 weeks at 7.5
major factor affecting the efficiency of thera- mg/kg with M23D AOs loaded onto ZM2
peutic drug delivery. nanoparticles. Immunohistochemical findings
showed 3% by manual count and 6% by semi-
Nanoparticles automated count of dystrophin-positive signal
in the heart. Dystrophin was also detectable,
In order to improve AO stability and enable the though faintly, in the heart of ZM2-M23D treat-
use of lower AOs concentrations in vivo, AO ed mice via Western blot. However, dystrophin
delivery using nanoparticles was developed was not detectable in the heart at 3 months
and demonstrated to be another promising post-injection as demonstrated in a follow-up
approach. Two classes of nanoparticles, T1 and study.
1209 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
Polymers Conclusion
Different series of polymers were investigated To summarize, with the exception of tricyclo-
for their ability to improve the delivery of AOs in DNAs, naked AOs are generally ineffective in
vitro and in vivo. The first class is a series of low treating the heart. Conjugation to CPPs, espe-
molecular weight polyethylenimine (LPEI) conju- cially arginine-rich peptides and the Pip series,
gated pluronic copolymers (PCMs) [174]. Am- appears to be a promising approach to improve
phiphilic pluronic, poly (ethylene oxide)-b-poly AO uptake and dystrophin restoration in cardi-
(propylene oxide)-b-poly (ethylene oxide) (PEO- ac tissue. However, the practicality of CPPs in
PPO-PEO triblock copolymer) has been used the clinical setting remains to be determined
widely as a pharmaceutical adjuvant and a free largely due to concerns over our complex
adjuvant for gene delivery. Depending on the immune system. Phage peptides and octagua-
molecular weight, hydrophilic-lipophilic balance nidine moieties were developed in an effort to
(HLB), and other characteristics of the pluron- reduce the immunogenicity of arginine-rich
ics, PCMs can have different AO delivery effi- peptides, however, they have been less suc-
ciencies and toxicities. In a study by Wang and cessful in rescuing dystrophin in systemic deliv-
colleagues, mdx mice were injected intrave- ery of antisense drugs to the heart. Other non-
nously with 0.4 mg of PCMs and 2 mg of PMOs covalent conjugation approaches such as the
targeting the boundary sequences of exon and use of nanoparticles, polymers, or ultrasound
intron 23 of mouse Dmd gene (PMOE23), or 2 and microbubbles are promising strategies to
mg of PMOE23 only as the control. Two weeks enhance the efficacy of AOs in cardiac tissue,
after injection, dystrophin was not detectable especially for charged antisense chemistries.
in cardiac muscle of control mice, while immu- With a growing number of AO-based approach-
nohistochemistry showed membrane-localized es being investigated, antisense therapy holds
dystrophin in more than 5% of cardiomyocytes the potential to improve the treatments for
in some areas of the heart from treated mice. DMD patients, with special focus on cardiac
Treatment also demonstrated no detectable aspects of the disease.
toxicity in muscles, liver, and kidneys after sys-
temic administration. Tricyclo-DNA clearly appears as a promising
chemistry for the antisense therapy of DMD-
Another class of amphiphilic polymers, termed associated cardiomyopathy due to its unique
Z polymers, is constructed from Tween 85 and pharmacological properties and high therapeu-
LPEI [175]. Based on preliminary results, two Z tic potential. Besides their ability to improve
polymers Z7 and Z8 were selected for systemic delivery and uptake into cardiac tissue, tcDNAs
injection. Dystrophic mdx mice were treated by can also cross the blood-brain barrier at low
IV injection with 1 mg Z7/1 mg PMO complex, or levels after systemic administration [139].
0.5 mg Z8/1 mg PMO conjugate, or 1 mg PMO Along with other distinctive properties, this out-
as a control. Dystrophin expression was not standing feature opens up more therapeutic
detected in the PMO-only group, however, options for other neuromuscular and neurode-
Z8-PMO treated mice demonstrated mem- generative disorders such as spinal muscular
brane-localized dystrophin-positive fibers in atrophy and Huntington’s disease [135].
some areas of the heart. No adverse effects Although the safety profile of tcDNAs has been
were observed in systemic delivery under the evaluated, observed toxicities were ascribed
experimental conditions. The mechanism/s by more to the phosphorothioate linkages rather
which amphiphilic polymers improve gene than the tcDNA chemistry itself [138]. More
transfer is/are not fully understood. Their hydro- extensive toxicological studies are recommend-
phobic interaction with cell membranes could ed to assess the full safety profile of this prom-
reduce membrane viscosity and facilitate the ising chemistry. Given its positive performance
entry of AOs [173]. Amphiphilic polymers could in the preclinical phase, the evaluation of tcD-
also prevent nonspecific binding of AOs to NAs in human clinical trials seems to be the
charged extracellular components, thus most logical next step. Similar to other chemis-
increasing the effective concentrations of ther- tries, the outcome essentially depends on how
apeutic drugs. well tcDNAs will be tolerated in patients.
1210 Am J Transl Res 2019;11(3):1202-1218Antisense therapy for DMD cardiomyopathy
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Disclosure of conflict of interest pectancy since 1967 and the impact of home
nocturnal ventilation. Neuromuscul Disord
None. 2002; 12: 926-929.
[13] Passamano L, Taglia A, Palladino A, Viggiano E,
Address correspondence to: Dr. Toshifumi Yokota, D’Ambrosio P, Scutifero M, Cecio MR, Torre V,
De Luca F, Picillo E, Paciello O, Piluso G, Nigro
Department of Medical Genetics, Faculty of
G, Politano L. Improvement of survival in Duch-
Medicine and Dentistry, University of Alberta, 8812- enne muscular dystrophy: retrospective analy-
112 St., Edmonton, AB T6G 2H7, Canada. Tel: sis of 835 patients. Acta Myol 2012; 31: 121-
+1-780-492-1102; E-mail: toshifum@ualberta.ca 125.
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