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Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges


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Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.
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Antisense Oligonucleotide-Mediated Exon Skipping for
Duchenne Muscular Dystrophy: Progress and Challenges
Virginia Arechavala-Gomeza, Karen Anthony, Jennifer Morgan, Francesco Muntoni
Dubowitz Neuromuscular Centre, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK
Abstract:Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular
disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF)
preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads
to the degeneration of muscle fibres, progressive weakness and premature death. Antisense
oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping
of targeted exons, restoring the ORF and the consequent production of a shorter but functional
dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD
and progress towards clinical application has been rapid. Less than a decade has passed between the
first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the
results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now
underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to
cardiac muscle remain significant challenges. Here we review the current status of AO-mediated
therapy for DMD, discussing the pre-clinical, clinical and regulatory hurdles and their possible
solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.
Keywords: Antisense oligonucleotides, clinical trials, duchenne muscular dystrophy, becker
muscular dystrophy, dystrophin, exon skipping, RNA therapy
Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is a
fatal, X-linked, neuromuscular disorder
that affects 1 in 3,500 newborn boys.
Patients are typically diagnosed as
toddlers; they develop progressive muscle
weakness and cardiomyopathy and lose
the ability to walk by their early teens.
Unless appropriate standards of care
(including non-invasive ventilation,
glucocorticoid and cardio-protective
treatment) are implemented, premature
death by cardiac or respiratory failure
occurs in the second decade of life (1-3).
DMD is caused by mutations in the DMD
gene that disrupt the open reading frame
(ORF) thus aborting the full translation of
its protein product, dystrophin (4, 5). The
DMD gene comprises 79 exons and the
majority (~65%) of mutations responsible
for DMD are out-of-frame deletions,
although duplications (~10%), small
mutations including non-sense and splice
site changes (~22%) and deep intronic
mutations (~3%) are also documented (6,
7). Some DMD deletions are more
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frequent than others and the gene has
two deletion hotspots (6): the most
commonly mutated region is exons 45–55
followed by exons 2–19.
Dystrophin is located underneath the
sarcolemma and connects the sub-
sarcolemmal cytoskeleton to the
extracellular matrix by binding N-
terminally to cytoskeletal F-actin and to
β–dystroglycan via a cysteine rich domain
near the C-terminus (8) (Top panel, Figure
1). It contains four main functional units:
an N-terminus, a central rod domain, a
cysteine rich domain and a C-terminal
domain. The central rod domain consists
of 24 spectrin-like repeats and four hinge
domains (9). Dystrophin interacts with
actin at both its N-terminus and via
spectrin-like repeats 11-17; the C-
terminus has also recently been shown to
allosterically affect actin binding (10). The
cysteine rich domain binds to β-
dystroglycan (BDG) (11-15) and the C-
terminal domain is required for binding to
syntrophin (16) and dystrobrevin (17).
These and other sarcolemmal proteins
such as the sarcoglycans are components
of the dystrophin associated glycoprotein
complex (DGC). Dystrophin and the DGC
play an important role in stabilising the
muscle fibre against the mechanical forces
of muscle contraction by providing a
shock-absorbing connection between the
cytoskeleton and the extracellular matrix.
Loss of dystrophin leads to disruption of
the complex, which results in
inflammation, increased intracellular
calcium influx, muscle degeneration and
replacement of muscle with adipo-fibrous
tissue (4). In addition, dystrophin plays a
role in signalling and is associated with
members of the stretch-activated calcium
channels; their mislocalisation and
dysfunction in dystrophic muscle
contributes to disease progression (18).
Spectrin repeats 16 and 17 within the
central rod domain, encoded by exons 42–
45, are also required for binding to
neuronal nitric oxide synthase (nNOS) (11,
12, 19). nNOS regulates the blood flow in
skeletal muscle (20); disruption of this
pathway may contribute to DMD
pathogenesis by inducing paradoxical
vasoconstriction during exercise (21).
Naturally-occurring dystrophin positive
“revertant fibres” (isolated or less
commonly small clusters of fibres strongly
positive for dystrophin) and “traces”
(fibres expressing very low levels of
dystrophin at the sarcolemma) occur in
more than 50% of the muscle biopsies of
DMD patients (22). Revertant fibres
represent a very small percentage of the
total fibres, in which somatic mutations or
stochastic alternative splicing events of
the dystrophin pre-mRNA lead to exon
skipping, the restoration of the ORF and
consequent expression of dystrophin (23,
24). Revertant dystrophins are correctly
localised to the sarcolemma and associate
with other DGC proteins, suggesting a
retained function (25, 26). Revertant
fibres have been well characterised in the
mouse model of DMD, the mdx mouse
(24, 27, 28). Whilst traces have not been
described in the mdx mouse, they are
present in approximately a third of DMD
patients (22) and may represent up to
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25% of the total muscle fibres (29). The
molecular mechanism of trace dystrophin
expression remains to be elucidated, but
it is thought to be at least in part different
from that of revertant fibres. For example,
traces can express different dystrophin
epitopes than the surrounding revertant
fibres (22).
Becker muscular dystrophy
Mutations in the DMD gene are also
responsible for a milder disorder, Becker
muscular dystrophy (BMD), a disease with
an extremely variable spectrum of
severity ranging from patients with
walking difficulties in their late teens or
early twenties, to the majority of
individuals in whom ambulation is
preserved into late adulthood and who
have an essentially normal lifespan (30).
DMD and BMD mutations differentially
affect the DMD gene: in DMD the
mutations disrupt the reading frame,
while mutations that cause BMD maintain
the ORF (31, 32) leading to the production
of an internally deleted dystrophin
protein. The size of the deletion does not
correlate with the severity of the disease,
as long as the reading frame rule is
maintained (33-40), and provided crucial
domains of dystrophin such as the β-
dystroglycan binding site are not removed
by the deletion. While the central and
distal rod domain is less vital for function
(35), (some patients missing these
domains only have very mild disease
manifestations such as myalgia and
muscle cramps, and mild weakness), in
frame deletions that affect the binding of
dystrophin to other proteins such as
cytoskeletal actin or β-dystroglycan result
in a severe phenotype (41, 42).
The existence of revertant fibres in DMD
and the occurrence of mildly affected
BMD individuals with in-frame deletions
suggest that it is feasible to modify
splicing by exon skipping (Figure 1) and
induce the production of functional
dystrophin in DMD patients, as long as
crucial domains of dystrophin are not
disrupted. Artificially restoring the ORF in
this way is thus an attractive therapeutic
strategy for DMD, as approximately 70%
of DMD patients have mutations
amenable to exon skipping (43).
Figure1 Exon skipping principle. (Next page) Upper panel:
Schematic representation of dystrophin mRNA (in-frame exons
are represented as square boxes, out-of-frame exons round or
arrow boxes). Normal splicing of these exons produces
dystrophin protein (pictured immediately below) retaining
functional protein-binding domains and correctly localised to
the sarcolemma (see section of control muscle stained with
anti-dystrophin antibody Dys2). Lower panel: representation of
dystrophin pre-mRNA highlighting the differences in splicing
between a Del48-50 DMD patient (left) and a Del48-51BMD
patient (right). The DMD deletion disrupts the open reading
frame (ORFs) which results in unstable mRNA and the absence
of functional dystrophin protein in muscle sections. In the BMD
patient the deletion maintains the ORF and generates the
production of an internally deleted dystrophin isoform that
retains the critical amino and carboxyl terminals and Cysteine -
rich domains. The ORF can be corrected by forced skipping of
exon 51 by directing antisense oligonucleotides to sequences
within exon 51 or to neighbouring intronic regions. Exon 51
skipping restores the ORF, generating a dystrophin equivalent
to that of the BMD patient. Insert table: Theoretical
applicability of single exon skipping in a series of DMD
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Table 1 Comparison between the two leading candidates for exon 51 skipping.SC = subcutaneous; IV = intravenous; IM =
Company Prosensa/GlaxoSmithKline AVI-BioPharma
2’O-methyl phosphorothioate
morpholino oligomer (PMO)
Backbone structure
Size and sequence
20 mer
30 mer
Delivery SC IV
Plasma protein binding
Backbone binds to serum
proteins No
Serum half life <4 h to 28 days (44, 45) 1.62 to 3.60 hours (46)
Max non-toxic dose in patients:
Proteinuria seen in all patients
at 6mg/kg (44) Not reached (46)
Max tested non-toxic dose in
mice: ?960mg/kg (47)
Max tested non-toxic dose in
primates: ?320mg/kg (47)
Orphan drug Yes Yes
Total number of patients 12 (44) 19 (46)
treatment Post-treatment
Maximum reported
dystrophin-positive fibres Not done 100% 5% 55%
Maximum dystrophin signal
intensity to control muscles by
immunofluorescence Not done 15.6% 11% 27%
Maximum dystrophin protein
level to control muscles by
western blotting Not done Not done 5% 18%
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Antisense oligonucleotide-mediated exon
Antisense oligonucleotides (AOs) targeted
to splicing elements represent the most
clinically advanced therapeutic tools
developed to induce dystrophin exon
skipping (48, 49). AOs are typically 20–30
nucleotides in length, and complementary
in sequence to regions of the pre-mRNA
transcript (50). While several AO
chemistries exist, the two AOs in clinical
development for DMD are 2’O-methyl
phoshophorothioate oligoribonucleotide
(2’OMe) and phosphorodiamidate
morpholino oligomers (PMO), (Table 1)
and (51, 52) for a detailed review. 2’OMe
AOs bind to albumin, showing high plasma
concentrations and long half-lives (45);
this might be an advantage as PK studies
indicate a longer persistence in blood
compared to PMO (up to 28 days as
opposed to less than 4 hours); however
binding to protein has been shown to
trigger activation of the immune system,
anaphylaxis, hypotension, or
antiarrhythmic effects in preclinical and
clinical studies (53). PMOs are not
metabolised and are resistant to
endonucleases (54); they are rapidly
eliminated from the bloodstream as they
are uncharged and do not bind serum
proteins, which is likely why they have not
been associated with the side effects
mentioned for the 2OMe clinical studies.
Both AOs have proven successful in pre-
clinical mouse models (55-58) and as far
as the PMO is concerned, also the more
severe dog model (59), in which systemic
delivery has resulted in dystrophin protein
production (60, 61) and physiological
improvement (58) of skeletal muscle.
Clinical progress
Both PMO (AVI-4658/eteplirsen) and
2’OMe (GSK-2402968) AOs targeting exon
51 (which will restore the ORF in the
largest group of DMD patients (13%))
have proven successful at inducing local
dystrophin expression in pivotal proof-of-
concept intramuscular clinical trials (62,
63). Recently, systemic studies using the
two different AO chemistries have been
completed (Table 2) (44, 46),
demonstrating that AO therapy for DMD is
indeed safe and well tolerated with no
significant drug-related adverse events.
Both studies reported significant
dystrophin restoration in a dose-
dependent manner as determined by
western blotting and
immunohistochemistry, with levels of
dystrophin approaching 20% of normal
levels in the PMO study.
The outcome of randomised placebo-
controlled studies of both eteplirsen and
GSK-2402968 is expected in 2012 and
further studies are planned. Table 2
summarises the design of both completed
and ongoing registered
studies correct at the time of publication.
In addition, AOs for exons 45, 52, 53 and
55 are undergoing pre-clinical
development by GSK whilst AVI
BioPharma is developing PMOs targeting
exons 45, 50 and 53. Plans to extend trials
of systemically-delivered AOs to non-
ambulant boys are also underway for
exon 51 skippable patients (both with
eteplirsen and GSK2402968).
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Study drug
trial register:
number: 2007-
(parent study:
Study design
controlled, dose-
Single dose
multiple Dose
double blind,
double blind
double blind
escalating dose,
open label
Number of patients
Target exon
Subcutaneous &
I.V (1.5, 5mg/kg)
0.09 and 0.9 mg
0.8 mg
0.5, 1, 2, 4, 10,
and 20 mg/kg
body weight
0.5, 2, 4 and 6
mg/kg body
30, 50
body weight
mg/kg body
3, 6
mg/kg body
6 mg/kg body
3, 6, 9 & 12 mg/kg
body weight
3, 6 mg/kg body
0.5, 1.5, 5, 8, 10,
12 mg/kg body
Frequency of
Weekly & twice
4 weeks
4 weeks
24 weeks
48 weeks
4 weeks
1 year
5 weeks
5 weeks
Primary outcome
Adverse events
positive fibers
6 minute walk
distance test
MRI ch
anges in
skeletal muscle
& dystrophin
Start Date
October 2007
January 2009
July 2011
October 2011
December 2010
July 2010
December 2009
Completion date
March 2009
December 2010
June 2012
April 2013
December 2012
November 2011
December 2012
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(Previous page) Table 2 Summary of completed
and on-going exon skipping clinical trials for DMD
IV = intravenous; IM = intramuscular
Current challenges
In-vitro optimisation of novel AOs
Systematic screening for AO targets has
already identified targets for most of the
79 dystrophin exons (64-66), but there is
variability in the processes used to design
and evaluate target AOs. For example,
variations in the cell type for in vitro
studies, transfection reagents, time of
evaluation and quantification of skipped
product (64, 67-69) make inter-study
comparisons difficult. Despite the fact
that bioinformatic tools (70-73) can
provide optimal target areas for AO
binding, and help rank AO sequences
according to their predicted bioactivity
(43, 65), empirical analysis in-vitro is
always necessary to confirm the suitability
of the sequence (69). Importantly,
restoration of dystrophin expression can
only be shown in differentiated myotubes
derived from patients’ cells as dystrophin
is only expressed in myotubes and not in
myoblasts. Ideally, cells from several
patients holding different amenable
deletions should be used to test the
efficacy of an AO, as the intronic
breakpoints differ between patients and
might affect splicing efficiency (74).
Primary myoblasts derived from DMD
muscle biopsies can be difficult to expand
in culture (75, 76) and the extent of
myogenic differentiation of DMD
myoblasts is often low (77, 78). Similar
levels of differentiation would be required
for quantitative comparison of the
efficacy of the same AO on cells from
patients with different mutations. In
order to improve the proliferative capacity
of human myoblasts so that large
numbers of cells are available for replicate
experiments, techniques to immortalise
human myoblasts have been developed
A less invasive alternative to the use of
muscle biopsies to prepare satellite cell-
derived myoblasts, are fibroblasts
prepared from a skin biopsy. Fibroblasts
can be induced to differentiate into
myotubes by forced expression of the
myogenic regulatory factor MyoD (80).
However, the levels of DMD transcripts in
myotubes derived from fibroblasts can be
low and the variable extent of myogenic
differentiation should be controlled for in
comparative experiments.Transgenic
mice, harboring the entire human DMD
locus, may be used to test antisense
oligonucleotides (64, 67, 81-83).
However, these mice also carry the mouse
dystrophin gene and have no skeletal
muscle pathology.
Outcome measures
A validated set of clinical outcome
measures for ambulant DMD patients is in
use in the ongoing phase II and III clinical
trials. Future trials on non-ambulant
patients pose a further challenge where
robust measurements of upper limb
strength and function in late disease stage
are required. While clinical outcome
measures are needed to demonstrate
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functional improvement, biochemical
outcome measures (BOMs) are required
to monitor AO efficacy. However, critical
differences in the methodology used
between the different research centres
are of concern. Standardised BOMs are
essential in order to reliably compare the
efficacy of the different chemistries and
dosing regimens. Specifically, the most
reliable methods for quantification of
both exon skipping and dystrophin
restoration must now be established
through initiatives such as the TREAT-
NMD registry of outcome measures for
neuromuscular disorders
( and from on-
going international collaborative studies
aiming to cross-validate standard
operating procedures. The outcomes of
these should be the standardisation of
methodology across centres that could be
presented to regulatory authorities as the
preferred BOMs in future clinical trials.
The efficacy of exon skipping is measured
at both the RNA and protein level. Nested
RT-PCR is traditionally used to assess and
quantify (semi-quantitatively) AO efficacy
at the RNA level (84, 85). To detect
transcripts using this method, it is
necessary to use up to 70 PCR cycles after
which linearity is lost and it is therefore
not possible to accurately quantify the
percentage of exon skipping. Thus several
quantitative methods are currently in
development such as qRT-PCR using highly
specific TaqMan assays for skipped and
total dystrophin targets. The advent of
digital PCR and micro fluidic technology
enables the high throughput analysis of
patient RNA. For example, exon skipping
could be assessed by measuring changes
in mRNA decay pre and post treatment
using TaqMan assays that cover all 79
dystrophin exons. Such a platform, the
FluiDMD, has recently been described
which simultaneously analyses 85 TaqMan
assays recognising 76 out of 78 DMD exon
junctions (86).
As the aim of AO-mediated exon skipping
is to restore dystrophin production,
reliable methods to quantify dystrophin
expression are vital. The presence of
dystrophin traces and revertant fibres in
DMD muscles (22) makes it essential to
compare treated muscles with a pre-
treatment biopsy of the same patient, in
order to accurately distinguish and
quantify AO-mediated dystrophin protein
production (87). The two most commonly
used methods are western blotting and
immunostaining (46, 87, 88).
Consideration should be given to the
antibodies to be used, which must be
sensitive, specific and have an epitope
appropriate for the dystrophin exons
retained following exon skipping.
As muscle biopsies are invasive and
sample a single muscle, there are
limitations in their use to monitor
response to therapy and efforts are being
made to identify non-invasive biomarkers
to monitor DMD disease progression.
Studies aimed at validating the role of
magnetic resonance imaging (89) and
spectroscopy as well as serum or urine
biomarkers such as small non coding RNAs
(such as miRNA) are currently underway
both in animal models and in clinical
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studies; it is hoped that these will reduce
the need for muscle biopsies (90).
Functionality of the de novo dystrophin
Whilst the primary outcome measure of
the completed systemic trials was clinical
safety, the GSK-2402968 study also
reported a modest improvement in the 6-
minute walk test which is encouraging
(44). From a biochemical perspective, data
from both the intramuscular and systemic
eteplirsen trials further indicate that the
internally deleted dystrophins generated
by exon skipping in different patients are
indeed functional, as they led to the
restoration of proteins of the DGC (46,
91). Additional evidence of functional
improvement is provided by a reduction in
cytotoxic T cells within treated muscle
biopsies (46); this is promising considering
that the pre-symptomatic induction of
inflammatory cascades and the invasion of
muscle by immune cells is one of the
earliest pathways induced in dystrophin
deficient muscle and is thought contribute
to DMD pathology (92, 93). However, the
possibility of an immunological reaction
both against revertant and novel
dystrophin epitopes remains a possibility
(94) and presents a new issue to address
in future clinical trials that will require the
assessment of any pre-existing
immunological response to dystrophin
epitopes in patients prior to their
inclusion in a clinical trial, as well as any
post-treatment response to the newly-
generated dystrophin protein (94).
Some mild or asymptomatic BMD patients
naturally express the dystrophin proteins
that we aim to produce by exon skipping
(32). A recent study correlated dystrophin
and dystrophin-associated protein
expression with disease severity in a
cohort of BMD patients (26). The amount
of dystrophin, nNOS and BDG correlated
to clinical severity and BMD patients with
deletions equivalent to those created by
exon 51 skipping have higher dystrophin
levels than either those with large multi-
exon deletions, or those harbouring exon
53 skippable deletions (26). These findings
demonstrate the therapeutic potential of
the protein that will be generated by exon
51 skipping trials whilst the functionality
of other dystrophins, especially those with
larger internal deletions, is less clear (43,
Variability of response
The completed AO-mediated exon
skipping clinical trials have revealed a high
degree of variability in patient response,
even between patients harbouring the
same deletions (46). These findings
suggest that the variability is unlikely to
be due to inter-patient differences in
stability of the resultant protein,
immunological response, or the
pharmacodynamics of the PMO (46).
However, it has been suggested (46) that
differences in the genetic background,
such as intronic deletion breakpoints,
differences in the efficiencies of mRNA
splicing, or differences in the vascular
access of the AO to individual muscles
may contribute to the variable response.
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An important future goal must be to
understand the mechanisms behind this
variability and why some patients respond
better to treatment than others.
Interestingly studies in the mdx mouse
with both PMO and 2’OMe and in the
GRMD dog using PMO have identified
similar variability in response, even in the
same animal, when different muscles
were studied (61, 98-100). This- indirectly-
points towards stochastic events involved
in delivering the AO to skeletal muscle
rather than a genetic difference, although
more studies are needed to elucidate the
mechanism responsible for the observed
variability and whether this variability may
be reduced after long-term treatment as
indicated by studies on the mdx mouse
Next generation AOs
Although extremely high-doses of PMO
without modification can induce
dystrophin rescue in mdx cardiac muscle
(101), unmodified AOs are largely
unsuccessful at inducing exon skipping in
the heart and they do not cross the blood
brain barrier (56, 58). This is important
given that cardiac complications are
observed in up to 90% of DMD patients
(102) and that 1/3 of DMD patients suffer
cognitive impairment related to the
deficiency of dystrophin in the brain (103,
104). One approach to improve AO
targeting to cardiac muscle is the direct
conjugation of cell penetrating peptides to
AOs which improves AO delivery to
skeletal (105-112) and cardiac mdx mouse
muscles (111, 113-115); however the
toxicology of these conjugates has yet to
be ascertained. The fact that the
dystrophin protein is thought to have a
long half-life should increase the
possibility of achieving and maintaining
therapeutically-relevant dystrophin
protein levels with weekly or longer
dosing intervals
Regulatory hurdles
The regulatory process for developing AOs
to skip other dystrophin exons is at
present cumbersome as each new AO is
considered a novel drug and requires the
full battery of genotoxicity, rodent and
non-human primate acute and chronic
toxicity studies (reviewed in (116)). This
stringent assessment of safety is of
paramount importance, considering that
there has been little experience in dosing
individuals with AOs at high doses and for
durations exceeding 1 year (and
theoretically for a lifelong therapy).
Nevertheless, the current studies have not
reported severe drug related adverse
events; in addition most of the toxicity
related to AOs derives not from the
individual sequences but from the chronic
chemical load which is therefore largely
backbone but not sequence specific. It is
hoped that the positive clinical experience
gained from the exon 51 skipping studies
and hopefully also from other exons will
allow us to gather additional information
so that in the future these compounds
could obtain class approval and follow a
more informed and streamlined
regulatory process.
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Preclinical and clinical studies using two
different chemistries have demonstrated
the potential of antisense oligonucleotide-
mediated DMD exon skipping to modify
the progression of DMD. If progress in
this field continues at the pace of the last
decade, treatment for common DMD
mutations may soon be feasible. If no
sequence-specific toxic effect is found,
treatment of rare mutations could follow
as regulatory hurdles are overcome.
Furthermore this approach could treat
nonsense mutations or other frame-
shifting mutations located in in-frame
exons that could be removed by skipping
a single exon. The clinical development of
next generation AOs that effectively
target cardiac as well as skeletal muscle
will provide a significant quality of life
improvement for patients.
Virginia Arechavala-Gomeza is supported
by a Health Innovation Challenge Grant
from the UK Department of Health and
the Wellcome Trust, Karen Anthony is
supported by the Association Francaise
contre les Myopathies, Jennifer Morgan is
supported by a Wellcome Trust University
Award and Francesco Muntoni is
supported by the Great Ormond Street
Hospital Children’s Charity.
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Author’s Pre-print copy
Please cite as Arechavala-Gomeza et al, Curr Gene Ther. 2012 Jun 1;12(3):152-60.
... By contrast, RNA-based drugs overcome many limitations: they can be easily produced in large and standardized fashion scale, making them cost-effective and extremely versatile as treatments, as they can modulate different steps of RNA biology. 3 The clinical importance of RNA therapeutics is highlighted by the number of molecules (16) presently in the clinic. In this review, we will focus on neurological disorders for which 11 FDA-approved RNA drugs are available, namely 10 ASOs, 3 3 short interfering RNA (siRNA) and 1 RNA aptamer. ...
... It is caused by frame shifting or nonsense mutations, or intragenic deletions or duplications in the DMD gene, encoding for dystrophin and located on the X chromosome. 16 DMD affects 1 in 3600-6000 newborn boys, who develop muscle degeneration and lose the capability to walk by their early teens. 16 Because DMD is caused by many different mutations in the DMD gene causing out-of-frame mRNA, 16 diverse ssASOs have been designed to treat DMD patients. ...
... 16 DMD affects 1 in 3600-6000 newborn boys, who develop muscle degeneration and lose the capability to walk by their early teens. 16 Because DMD is caused by many different mutations in the DMD gene causing out-of-frame mRNA, 16 diverse ssASOs have been designed to treat DMD patients. 16 Here, a shorter but functional protein can be restored by skipping one or more exons, restoring in-frame sequence. ...
Introduction Ribonucleic acid (RNA) therapeutics are a new class of drugs whose importance is highlighted by the growing number of molecules in the clinic. Sources of data We focus on RNA therapeutics for neurogenetic disorders, which are broadly defined as diseases with a genetic background and with at least one clinical sign affecting the nervous system. A systematic search identified 14 RNA drugs approved by FDA and many others in development. Areas of agreement The field of RNA therapeutics is changing the therapeutic scenario across many disorders. Areas of controversy Despite its recent successes, RNA therapeutics encountered several hurdles and some clinical failures. Delivery to the brain represents the biggest challenge. Growing points The many advantages of RNA drugs make the development of these technologies a worthwhile investment. Areas timely for developing research Clinical failures stress the importance of implementing clinical trial design and optimizing RNA molecules to hold the promise of revolutionizing the treatment of human diseases.
... AONs are currently the largest modality of RNA-based therapeutics. Their rational design, chemistry and usage in cell, animal and clinical studies has been extensively reviewed elsewhere [4][5][6]. Briefly, AONs are short sequences of deoxynucleotides or deoxyribonucleotides which have been chemically modified to improve stability. The choice of chemistry largely depends on the desired application, all clinically approved AONs to date are either phosphorodiamidate morpholino oligomers (PMOs) or 2′-O-methoxyethyl (2ʹMOE) oligomers with a phosphorothioate (PS) backbone (2ʹMOE-PS) (Figure 3). ...
... In contrast, the parallel development of ssAONs for DMD utilized an exon skipping mechanism [6]. Duchenne is the most common type of muscular dystrophy and is caused by frame-shifting mutations in the DMD gene that prevent the full translation of its protein product, dystrophin [26]. ...
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RNA-based therapeutics have entered the mainstream with seemingly limitless possibilities to treat all categories of neurological disease. Here, common RNA-based drug modalities such as antisense oligonucleotides, small interfering RNAs, RNA aptamers, RNA-based vaccines and mRNA drugs are reviewed highlighting their current and potential applications. Rapid progress has been made across rare genetic diseases and neurodegenerative disorders, but safe and effective delivery to the brain remains a significant challenge for many applications. The advent of individualized RNA-based therapies for ultra-rare diseases is discussed against the backdrop of the emergence of this field into more common conditions such as Alzheimer's disease and ischaemic stroke. There remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system; coupled with the accelerated development expected over the next decade, the true potential of RNA-based therapeutics to transform the therapeutic landscape in neurology remains to be uncovered.
... Several antisense oligonucleotides (ASOs) that induce exon skipping in the DMD pre-mRNA have been approved as therapies for DMD (11)(12)(13)(14)(15). However, the efficacy of currently approved therapeutic approaches using systemic administration of exon skipping ASO with phosphorodiamidate morpholino oligomer (PMO) chemistry is limited by poor muscle delivery due to inefficient cellular uptake and rapid renal clearance (16)(17)(18). Thus, there is a need for new strategies to efficiently deliver exon-skipping oligonucleotides to muscle in patients with DMD. ...
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Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.
... This gene encodes the dystrophin protein that plays a key role in strengthening of muscle fibers. Antisense RNA-mediated gene therapy has shown encouraging results for treatment of DMD but lacks efficiency [122,123]. In the dmd gene, the exon 45-55 region is a known mutational hotspot and has been targeted extensively [124][125][126]. ...
A single gene mutation can cause a number of human diseases that affect quality of life. Until the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems, it was challenging to correct a gene mutation to avoid disease by reverting phenotypes. The advent of CRISPR technology has changed the field of gene editing, given its simplicity and intrinsic programmability, surpassing the limitations of both zinc-finger nuclease and transcription activator-like effector nuclease and becoming the method of choice for therapeutic gene editing by overcoming the bottlenecks of conventional gene-editing techniques. Currently, there is no commercially available medicinal cure to correct a gene mutation that corrects and reverses the abnormality of a gene’s function. Devising reprogramming strategies for faithful recapitulation of normal phenotypes is a crucial aspect for directing the reprogrammed cells toward clinical trials. The CRISPR-Cas9 system has been promising as a tool for correcting gene mutations in maladies including blood disorders and muscular degeneration as well as neurological, cardiovascular, renal, genetic, stem cell, and optical diseases. In this review, we highlight recent developments and utilization of the CRISPR-Cas9 system in correcting or generating gene mutations to create model organisms to develop deeper insights into diseases, rescue normal gene functionality, and curb the progression of a disease.
... All approved drugs are mutation specific and designed to rescue specific patient mutations only present, respectively, in 13% (ataluren), 13% (eteplirsen) and 8% of DMD patients 8 (golodirsen, viltolarsen and casimersen). It is therefore important to assess exon-skipping strategies targeting other DMD exons 9 and therapies that may benefit all DMD and BMD patients, independent of their mutations. One such potential therapy is gene transfer: several trials are ongoing testing different drugs (SGT-001, SRP-9001 or PF-06939926) that include mini or micro-dystrophins in adeno-associated viruses driven by different promoters. ...
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Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.
... While alternative splicing is regulated by exogenously synthesized oligo RNAs or endogenous natural antisense transcripts, greater target site specificity occurs due to sequence guided complementation. The first application of antisense RNA in clinic was for the treatment of patients with spinal muscular atrophy [125][126][127]. This should also be a promising therapeutic strategy for delivering splice-switching oligomers to lymphoid cells for splicing-associated immune diseases by correcting mis-splicing or altering the balance of different splice isoforms. ...
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The human immune response is a complex process that responds to numerous exogenous antigens in preventing infection by microorganisms, as well as to endogenous components in the surveillance of tumors and autoimmune diseases, and a great number of molecules are necessary to carry the functional complexity of immune activity. Alternative splicing of pre-mRNA plays an important role in immune cell development and regulation of immune activity through yielding diverse transcriptional isoforms to supplement the function of limited genes associated with the immune reaction. In addition, multiple factors have been identified as being involved in the control of alternative splicing at the cis, trans, or co-transcriptional level, and the aberrant splicing of RNA leads to the abnormal modulation of immune activity in infections, immune diseases, and tumors. In this review, we summarize the recent discoveries on the generation of immune-associated alternative splice variants, clinical disorders, and possible regulatory mechanisms. We also discuss the immune responses to the neoantigens produced by alternative splicing, and finally, we issue some alternative splicing and immunity correlated questions based on our knowledge.
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The conversion of fibroblasts into myogenic cells is a powerful tool to both develop and test therapeutic strategies and to perform in-depth investigations of neuromuscular disorders, avoiding the need for muscle biopsies. We developed an easy, reproducible, and high-efficiency lentivirus-mediated transdifferentiation protocol, that can be used to convert healthy donor fibroblasts and a promising new cellular model, urinary stem cells (USCs), into myoblasts, that can be further differentiated into multinucleated myotubes in vitro. Transcriptome and proteome profiling of specific muscle markers (desmin, myosin, dystrophin) was performed to characterize both the myoblasts and myotubes derived from each cell type and to test the transdifferentiation-inducing capacity of MYOD1 in fibroblasts and USCs. Specifically, the Duchenne muscular dystrophy (DMD) transcripts and proteins, including both the full-length Dp427 and the short Dp71 isoform, were evaluated. The protocol was firstly developed in healthy donor fibroblasts and USCs and then used to convert DMD patients' fibroblasts, with the aim of testing the efficacy of an antisense drug in vitro. Technical issues, limitations, and problems are explained and discussed. We demonstrate that MyoD-induced-fibroblasts and USCs are a useful in vitro model of myogenic cells to investigate possible therapies for neuromuscular diseases.
Rare diseases are most often caused by inherited genetic disorders that, after translation, will result in a protein with altered function. Decreased protein stability is the most frequent mechanism associated with a congenital pathogenic missense mutation and it implies the destabilization of the folded conformation in favor of unfolded or misfolded states. In the cellular context and when experimental data is available, a mutant protein with altered thermodynamic stability often also results in impaired homeostasis, with the deleterious accumulation of protein aggregates, metabolites and/or metabolic by‐products. In the last decades a significant effort has enabled the characterization of rare diseases associated to protein stability defects and triggered the development of innovative therapeutic intervention lines, say, the use of pharmacological chaperones to correct the intracellular impaired homeostasis. In here, we review the current knowledge on rare diseases caused by reduced protein stability, paying special attention to the thermodynamic aspects of the protein destabilization, also focusing on some examples where pharmacological chaperones are being tested. This article is protected by copyright. All rights reserved
Background : Muscular dystrophy (MD) is a group of multiple muscle diseases, which causes severely impaired motor ability, degeneration and dysfunctions in the musculoskeletal system, respiratory failure and feeding difficulties. LAMA2-related MD is caused by pathogenic variants in the LAMA2 gene, encoding laminin a2 chain, a component of the skeletal muscle extracellular matrix protein laminin-α2β1γ1. Here, we performed clinical examination and molecular genetic analysis in a patient with congenital MD (CMD), and autism-like phenotype. Methods : In this study we performed whole exome sequencing (WES) to find possible genetic etiology of CMD in an Iranian non-consanguineous patient. The pathogenicity of the variants was assessed using various Bioinformatics tools. American College of Medical Genetics and Genomics (ACMG) guidelines were used to interpret the variant and Sanger sequencing in the patient and her family was applied for the confirmation of the variant. Results : WES results showed a novel frameshift homozygous variant (p.Tyr1313LeufsTer4) in the LAMA2 gene leading to the CMD phenotype. This variant resides in a highly conserved region and was found to be co-segregating in the family. It fulfils the criteria of being pathogenic. Conclusion : Here, we successfully identified a novel LAMA2 pathogenic variant in an Iranian patient suffering from CMD and autism using WES. Identification of disease-causing variant in autosomal recessive disorders such as CMD can be useful in genetic counseling, prenatal diagnosis, and predicting prognosis of the disease.
Oligonucleotide RNA therapeutics are manufactured by a controlled and well-established chemical process and their sizes typically reach from 5000 to 20,000 or more Daltons. They are viewed by regulatory agencies as “large small molecules” as they predominantly are manufactured like new chemical entities but also share properties with biologics and therefore fall in a regulatory gray area between small molecule drugs and biologics. Together with the fact that they are a recently introduced young therapeutic modality, some sections of the ICH (International Conference for Harmonization) guidelines, like Q3A/B and Q6B, have so far excluded oligonucleotide therapeutics in their preambles. Nevertheless, with the recent approval of over a dozen of oligonucleotide-based therapeutics, more guidance is needed. This publication makes an attempt to guide a CMC chemist or anyone interested in oligonucleotide therapeutic development to learn how to navigate their program and to be more prepared in answering CMC and in particular impurity-related questions by the regulatory agencies.
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The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, α1, β1, and β2. Based on their domain organization and association with neuronal nitric oxide synthase, syntrophins are thought to function as modular adapters that recruit signaling proteins to the membrane via association with the dystrophin complex. Using sequences derived from a new mouse β1-syntrophin cDNA, and previously isolated cDNAs for α1- and β2-syntrophins, we prepared isoform-specific antibodies to study the expression, skeletal muscle localization, and dystrophin family association of all three syntrophins. Most tissues express multiple syntrophin isoforms. In mouse gastrocnemius skeletal muscle, α1- and β1-syntrophin are concentrated at the neuromuscular junction but are also present on the extrasynaptic sarcolemma. β1-syntrophin is restricted to fast-twitch muscle fibers, the first fibers to degenerate in Duchenne muscular dystrophy. β2-syntrophin is largely restricted to the neuromuscular junction. The sarcolemmal distribution of α1- and β1-syntrophins suggests association with dystrophin and dystrobrevin, whereas all three syntrophins could potentially associate with utrophin at the neuromuscular junction. Utrophin complexes immunoisolated from skeletal muscle are highly enriched in β1- and β2-syntrophins, while dystrophin complexes contain mostly α1- and β1-syntrophins. Dystrobrevin complexes contain dystrophin and α1- and β1-syntrophins. From these results, we propose a model in which a dystrophin–dystrobrevin complex is associated with two syntrophins. Since individual syntrophins do not have intrinsic binding specificity for dystrophin, dystrobrevin, or utrophin, the observed preferential pairing of syntrophins must depend on extrinsic regulatory mechanisms.
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Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains ~1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model.Keywords: antisense oligonucleotides; digital array; duchenne muscular dystrophy; exon skipping; transcript quantification
Antisense oligonucleotides (AONs) are typically applied as targeted downregulators of mRNA translation. This action is achieved by base pairing of specific 2-deoxyoligonucleotides with the targeted mRNA, a process that elicits destruction of the mRNA by RNase H, an enzyme that catalyzes RNA breakdown in an RNA/DNA duplex [12]. In recent years, the manipulation of splicing has been accomplished with chemically modified AONs that do not activate RNase H, thereby redirecting aberrant or alternative splicing rather than causing destruction of the targeted pre-mRNA, thus upregulating the synthesis of correct and/or therapeutic gene products. Benefits of such approach include possible use as a method to determine function of gene isoforms, positive readout assay for antisense activity, and most importantly, therapeutic potential for genetic diseases and a variety of other disorders that can be addressed by manipulation of alternative splicing of specific genes. In this chapter we will review pertinent literature since 2001, the time of publication of the previous edition of this volume, to present.
We have correlated a detailed clinical assessment of 67 patients with proven Becker muscular dystrophy with the results from genetic and protein analyses. There was an overall deletion frequency of 80%, rising to 92.6% in the large group of patients defined on clinical grounds as being of typically mild severity. The deletions in this group were all clustered in the region of the gene between exons 45 and 59; the most common deletion was of exons 45–47 and all but one started at exon 45. No similar deletions were seen in the patients with more severe disease, in whom the diverse genetic defects included a duplication and a very large deletion. Dystrophin patterns in the typical group were also very characteristic, and in both groups were as predicted from the genetic defect, the size of deletions being inversely proportional to the size of the protein produced.
Many features of dystrophin-deficient muscle pathology are not clearly related to the loss of mechanical support of the muscle membrane by dystrophin. In the present review, evidence that supports a role for the immune system in promoting the pathology of dystrophinopathy is presented. The findings summarized here indicate that specific, cellular immune responses by cytotoxic T-lymphocytes and helper T-lymphocytes contribute to muscle pathology in dystrophin-deficient muscle, and that removal of specific lymphoid cell populations can reduce muscle pathology. In addition, innate immune responses may also promote dystrophinopathies by the tremendous infiltration of myeloid cell populations into the dystrophic muscle. Loss of normal redox homeostasis by dystrophin-deficient muscle may increase its sensitivity to free radical-mediated damage by myeloid cells. Collectively, the observations presented here suggest that the contribution of the immune system to dystrophinopathies may be significant, and that therapeutic approaches based upon immune interventions may ameliorate the pathological progression of dystrophin deficiency.
Antisense-mediated splicing modulation of premessenger RNA represents a novel therapeutic strategy for several types of pathologies such as genetic disorders, cancers, and infectious diseases. Antisense oligonucleotides designed to bind to specific mRNA molecules have been actively developed for more than 20 years as a form of molecular medicine to modulate splicing patterns or inhibit protein translation. More recently, small nuclear RNA such as U7 or U1 small nuclear RNA have been used to carry antisense sequences, offering the advantage of long-term effect when delivered to cells using viral vectors. We have previously demonstrated the therapeutic potential of U7snRNA targeting dystrophin mRNA as a treatment for Duchenne muscular dystrophy. In particular, we showed that bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51, and thus restore dystrophin expression in DMD patients cells to near wild-type levels. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications. Here, we outline the design of these U7snRNA constructs to achieve efficient exon-skipping and describe methods to evaluate the efficacy of such U7snRNA constructs in vitro using the dystrophin gene as an example.