doi:10.1093/brain/awq002
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BRAIN
A JOURNAL OF NEUROLOGY
REVIEW ARTICLE
RNA-targeted splice-correction therapy for
neuromuscular disease
Matthew J. A. Wood,1 Michael J. Gait2 and Haifang Yin1
1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK
2 Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 0QH, UK
Correspondence to: Dr Matthew J. A. Wood,
Department of Physiology,
Anatomy and Genetics,
University of Oxford,
South Parks Road
Oxford OX1 3QX, UK
E-mail: [email protected]
Splice-modulation therapy, whereby molecular manipulation of premessenger RNA splicing is engineered to yield genetic correction, is a promising novel therapy for genetic diseases of muscle and nerve—the prototypical example being Duchenne
muscular dystrophy. Duchenne muscular dystrophy is the most common childhood genetic disease, affecting one in 3500
newborn boys, causing progressive muscle weakness, heart and respiratory failure and premature death. No cure exists for
this disease and a number of promising new molecular therapies are being intensively studied. Duchenne muscular dystrophy
arises due to mutations that disrupt the open-reading-frame in the DMD gene leading to the absence of the essential muscle
protein dystrophin. Of all novel molecular interventions currently being investigated for Duchenne muscular dystrophy, perhaps
the most promising method aiming to restore dystrophin expression to diseased cells is known as ‘exon skipping’ or
splice-modulation, whereby antisense oligonucleotides eliminate the deleterious effects of DMD mutations by modulating
dystrophin pre-messenger RNA splicing, such that functional dystrophin protein is produced. Recently this method was
shown to be promising and safe in clinical trials both in The Netherlands and the UK. These trials studied direct antisense
oligonucleotide injections into single peripheral lower limb muscles, whereas a viable therapy will need antisense oligonucleotides to be delivered systemically to all muscles, most critically to the heart, and ultimately to all other affected tissues including
brain. There has also been considerable progress in understanding how such splice-correction methods could be applied to the
treatment of related neuromuscular diseases, including spinal muscular atrophy and myotonic dystrophy, where defects of
splicing or alternative splicing are closely related to the disease mechanism.
Keywords: RNA splicing; antisense oligonucleotide; exon-skipping; dystrophin
Abbreviations: BMD = Becker muscular dystrophy; DM1 = type 1 myotonic dystrophy; DM2 = type 2 myotonic dystrophy;
DMD = Duchenne muscular dystrophy; pre-mRNA = pre-messenger RNA; PMO = morpholino phosphorodiamidate; SMA = spinal
muscular atrophy; SMN = survival motoneuron; snRNA = small nuclear RNA
Introduction
RNA plays a central role in the regulation of gene expression and
cell physiology (Sharp, 2009), hence the importance of both
precisely controlled RNA processing and the advent of new
types of molecular therapies that target RNA. A critical step in
the processing of pre-messenger RNA (pre-mRNA) in (eukaryotic)
mammalian cells, including brain and muscle, involves intron
Received October 9, 2009. Revised December 11, 2009. Accepted December 12, 2009. Advance Access publication February 11, 2010
ß The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
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excision—known as pre-mRNA splicing—to yield a mature mRNA
that, after editing, will include coding elements and signals appropriate for translation into functional protein (Crick, 1979). The
natural complexity of pre-mRNA splicing also allows the exclusion
(or inclusion) of specific exons by a process known as alternative
splicing (Matlin et al., 2005). While there is a surprisingly small
amount of diversity in genome size between organisms of widely
differing complexity, the importance of alternative splicing lies in
its contribution to the generation of proteomic diversity. In an
extreme example of the amplification of proteomic diversity possible through alternative splicing, the Drosophila ‘Dscam’ gene,
which encodes a cell surface axon guidance protein, generates
as many as 38 016 alternatively spliced isoforms (Schmucker and
Flanagan, 2004). Moreover, alternative splicing is now known to
be the norm rather than the exception within the human genome,
with up to 70% of genes generating alternatively spliced products
M. J. A. Wood et al.
(Modrek and Lee, 2002; Hertel, 2008). The control of alternative
splicing events involving the definition of exon/intron boundaries
through spliceosome and small nuclear RNA (snRNA) recognition
of pre-mRNA signatures is complex but critical for cell function
(Fig. 1). Perhaps unsurprisingly, alternative splicing is known to be
especially prominent in the mammalian nervous system, where the
resulting rich proteomic diversity reflects the complexity of cellular
processes and interactions (Blencowe, 2006). Recently, through
the development of increasingly powerful genome-wide mapping
methods it is now possible to reveal specific splicing factor
(protein)–RNA interaction maps across whole brain (Licatalosi
et al., 2008). Taken together, it is therefore not unexpected that
an increasing number of neurological and neuromuscular disorders
are being discovered to have direct or indirect links to defects
in constitutive or alternative splicing (Table 1); perhaps the best
characterized examples being Duchenne muscular dystrophy
Figure 1 Alternative splicing: regulation and the generation of proteomic diversity. Alternative splicing generates diverse protein isoforms.
Splicing is initiated by recognition of the 5’ splice site (GU) by the U1 snRNP (small nuclear ribonucleoprotein) and 3’ splice site including
the branch point, polypyrimidine tract and AG by the U2 snRNP/U2AF splicing factor complex. Upon binding of the other splicing factors,
a lariat is formed between a 5’ splice site and a branch point. A rearrangement then occurs that removes the lariat and ligates the exons
together. The presence of splice enhancers and silencers, which affect the recognition of the splice sites by the respective factors, can result
in a number of alternatively spliced variants arising from a single pre-mRNA transcript, generating a range of different protein isoforms.
Four examples of this are shown (arrowed numbers 1–4 in the left, middle-left, middle-right and right hand panels), including: alternate
inclusion of exons within the final mature mRNA transcript (1 versus 2); usage of different 5’ and 3’ splice sites leading to alternate exon
usage (2 versus 3) and exclusion of exons (4). These and other modes of pre-mRNA usage can result in alternate transcription start sites,
coding region usage, stop codon introduction and different 5’ and 3’ untranslated regions, all of which can contribute to mature mRNA
and thus proteomic diversity.
CGG repeat expansions in
FMR1 gene
MAPT gene mutations affecting
splicing enhancers or silencers
Fragile X and Fragile X-associated
tremor/ataxia syndrome
Frontotemporal dementia and
parkinsonism linked to
chromosome 17
DMD and BMD
CCTG expansion mutation in intron
1 of the zinc finger
protein 9 (ZNF9) gene
Mutations in NF1 gene
DM2
Deletion/mutation of the
SMN1 gene
Triplet repeat expansions in
coding regions of relevant genes
SMA
CUGBP1 = CUG binding protein 1; MBNL1 = muscleblind-like 1.
Spinocerebellar ataxias SCA2,
SCA8, SCA10 and SCA12
Defective HBII-52 snRNA
splicing regulator
Prader–Willi syndrome
Neurofibromatosis type 1
DM1
Non-coding mutations of
hepatocyte growth factor (HGF)
CUG expansion in the 3’untranslated
region of DMPK gene
Non-syndromic hearing loss
DMD gene deletions and mutations
Defects and mutations in TAR
DNA-binding protein-43 (TDP-43) and
FUS/TLS DNA/RNA-binding protein
Mutations within the ATM gene
Amyotrophic lateral sclerosis
Ataxia telangiectasia
Aetiology
Disorder
Table 1 Neuromuscular disorders linked to mRNA splicing
Dysregulated activity of MBNL1
and CUGBP1 splicing regulators,
leading to widespread spliceopathy
and defective expression of adult
splice variants in skeletal muscle
Misregulated activity of CUGBP1
RNA splicing factors and dysregulation
processing of target pre-mRNAs
Aberrant pre-mRNA splicing
and exon skipping
Leads to dysregulated alternative
splicing of serotonin receptor
2C pre-mRNA
SMN1 loss-of-function;
modulated by SMN2 copy number
RNA gain-of-function leading to splicing
dysregulation including altered activities of
CUGBP1 and MBNL1 splicing factors
Activation of cryptic splice sites
and affects on RNA structure
causing defective ATM
pre-mRNA splicing
Gain-of-function RNA effect
via sequestration of
RNA-splicing factors
Altered alternative splicing
of microtubule-associated
protein tau exon 10
DMD gene loss-of-function with
infrequent splicing defects
leading to phenotypic
heterogeneity
Splicing dysregulation
Transcriptional and
RNA splicing defects
Mechanism
Cartegni et al., 2006;
Talbot and Davies, 2008
Moseley et al., 2006;
Ranum and Cooper, 2006; Daughters et al., 2009
Kishore and Stamm, 2006
Ars et al., 2000
Margolis et al., 2006;
Osborne and Thornton, 2006
Osborne and Thornton, 2006;
Ranum and Cooper, 2006
Schultz et al., 2009
D’Souza et al., 1999;
Grover et al., 1999;
Andreadis, 2005
Ahn and Kunkel, 1993;
Muntoni et al., 2003
Jin et al., 2003;
Ranum and Cooper, 2006
Licatalosi et al., 2008;
Lagier-Tourenne and Cleveland, 2009;
Zhang et al., 2009
Teraoka et al., 1999;
Pagani et al., 2002;
Buratti et al., 2007
References
Neuromuscular disease splice therapy
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(DMD), spinal muscular atrophy (SMA) and the myotonic dystrophies. In recent years, a new class of molecular therapies targeting
RNA has arisen, including therapies aimed at correcting defects
arising in pre-mRNA splicing—the so-called ‘splice-correction’ or
‘splice-modulation’ therapies. Here we review the recent progress
and challenges in the development of such splice-modulation
therapies for neuromuscular disease, focusing primarily on DMD
where encouraging Phase I clinical trials have recently been
completed.
DMD and splice-modulation
therapy
DMD and its milder allelic variant Becker muscular dystrophy
(BMD) are X-linked disorders that arise from mutations, typically
large deletions, in the DMD gene—the largest gene in the human
genome (Muntoni et al., 2003). In the case of DMD, deletions
(65%), but also duplications, point mutations or other smaller
gene rearrangements affect alternative pre-mRNA splicing to
generate an out-of-frame DMD transcript, leading to aberrant
pre-mRNA translation and consequently an absence of the essential muscle protein dystrophin. Dystrophin, first characterized in
1987 (Hoffman et al., 1987), is an integral molecular constituent
of the dystrophin-glycoprotein complex, a large multi-component
membrane complex that provides a mechanical and signalling link
between the actin cytoskeleton in muscle and the extracellular
matrix (Davies and Nowak, 2006). The absence of dystrophin renders muscle cells susceptible to stretch-induced damage, increased
intracellular calcium influx, and is followed by muscle fibre degeneration. Patients with DMD suffer from severe, progressive muscle
wasting leading to loss of ambulation and respiratory weakness.
Cardiac involvement is found in up to 90% of DMD patients and
a large number of patients develop dilated cardiomyopathy leading to cardiac death (Bushby et al., 2003). In contrast, BMD arises
from mutations/deletions in which the DMD pre-mRNA transcript
is maintained in-frame. As a result, pre-mRNA processing yields
a truncated dystrophin protein lacking domains corresponding to
the exonic sequences affected by the mutation. Thus, BMD is in
general much less severe a disorder than DMD, given that dystrophin protein is present, but there is considerable heterogeneity due
to the fact that different mutations result in the elimination of
distinct functional domains within the resulting dystrophin protein
(Aartsma-Rus et al., 2006a)—some more critical and essential for
function than others.
While glucocorticosteroid therapy is currently the gold standard
treatment for DMD, bringing about a modest increase in life
expectancy (Bushby et al., 2004), the most promising treatment
strategies for DMD aim to restore expression of dystrophin protein, thus targeting the primary defect. Several promising new
therapeutic methods currently exist (van Deutekom and van
Ommen, 2003). These include gene replacement therapy using
adeno-associated virus transduction. This approach presents
major challenges including the very high viral titres required for
systemic gene delivery, the size and complexity of the DMD gene
which currently precludes its packaging as an entire functional unit
M. J. A. Wood et al.
within the adeno-associated virus genome, and immunological
concerns. Nevertheless gene therapy is in early stage clinical
trials in the USA. A second method relates to up-regulation of
the closely related gene homologue utrophin, which has been
shown experimentally to compensate for the loss of dystrophin
function (Tinsley et al., 1996). Progress has been made to identify
small molecules capable of up-regulating utrophin expression
(Hirst et al., 2005) and it is anticipated that a lead compound
will enter clinical trials in 2010. A third therapeutic approach
applicable to point mutations in the DMD gene that introduce
premature termination codons (510% of all DMD patients),
is afforded by drugs that permit translational read-through of
such mutations. The prototype in this class is the small molecule
drug PTC124 (AtalurenÕ ), identified by high-throughput screening, which has shown encouraging safety and tolerability in Phase
I and IIa clinical studies (Welch et al., 2007). Perhaps most promising of all novel molecular interventions for DMD at present
is dystrophin splice-modulation, whereby short antisense oligonucleotides are utilized to manipulate processing of the DMD
pre-mRNA to restore the DMD open reading frame and yield
the production of de novo functional dystrophin protein
(Aartsma-Rus and van Ommen, 2007).
Molecular basis of antisense
oligonucleotide
splice-modulation for DMD
The theoretical basis for the development of splice-modulation
therapies for DMD is threefold. First, the idea that antisense oligonucleotides could be directed to modulate splicing of pre-mRNA
transcripts arose from seminal observations on the b-globin gene
associated with b-thalassaemia (Dominski and Kole, 1993).
Secondly, the existence of so-called revertant fibres, individual
dystrophin-positive fibres, found in at least 50% of all DMD
patients, indicated the presence of an intrinsic alternative splicing
mechanism that could, albeit erratically and at low efficiency, successfully restore dystrophin protein expression within individual
myocytes (Lu et al., 2000). Finally, the internally shortened
dystrophin proteins characteristically found in BMD patients are
capable of function, since they contain the critical carboxy- and
amino-terminal domains of the protein, as demonstrated in several
BMD families with in-frame deletions of, for example, exons 32–
44, 48–51 or 48–53 (Melis et al., 1998), and which as a result
display no clinical myopathy. This theoretical foundation has led to
the investigation of methods for splicing modulation of the DMD
gene over the last decade. Typically, effective antisense oligonucleotide-mediated splice-modulation of the DMD pre-mRNA
requires the elimination via exon skipping of specific DMD exons
and their flanking regions to yield an in-frame but internally truncated transcript capable of producing functional dystrophin protein
(Fig. 2). Effective exon skipping antisense oligonucleotides are
short, typically 20–30 nucleotides in length, and homologous in
sequence to regions of the pre-mRNA transcript at or in close
proximity to the relevant DMD exon targeted for skipping. Such
target sequences most commonly correspond to motifs or other
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Figure 2 Normal processing and antisense oligonucleotide-mediated splice-modulation of the human DMD gene. (A) Normal pre-mRNA
splicing in the human DMD gene within the ‘hotspot’ region of exons 48–52, showing successful splicing and the generation of dystrophin
protein comprising the essential functional domains at the N- and C-termini and including the cysteine-rich domain. Deletions of DMD
exons 50 or 49 and 50 are also shown. These deletions in the human DMD gene disrupt the genomic reading frame leading to an unstable,
non-functional dystrophin protein and hence disease due to dystrophin loss-of-function. (B) Antisense oligonucleotide-mediated exon
skipping for DMD. An antisense oligonucleotide directed towards DMD exon 51 can induce effective exclusion (skipping) of exon 51 and
restore the transcript reading-frame (as shown by the schematic exon ‘fit’), therefore generating a truncated (shortened rod domain) but
partly functional dystrophin protein. (C) Antisense oligonucleotide-mediated multi-exon skipping for DMD. In DMD patients bearing for
example a nonsense point-mutation in exon 51, skipping of more than one exon will be required to establish an in-frame DMD transcript.
By skipping both exons 50 and 51, in this case using a cocktail of antisense oligonucleotides directed to these exonic regions, a shortened
but again partly functional dystrophin protein can be produced. (D) Limitations to the exon skipping approach for DMD. Antisense
oligonucleotide-mediated exon skipping will not be applicable when the mutations directly disrupt critical functional dystrophin protein
domains (e.g. exon 66 in the distal cysteine-rich domain), since effective splice-modulation of these pre-mRNA defects will not yield
functional protein. DAPC = dystrophin associated protein complex.
sites within the target exon (e.g. exonic splicing enhancer or other
sites) (Aartsma-Rus et al., 2005, 2009b; Wilton et al., 2007) but
may also target neighbouring intron/exon boundaries (splice
donor or splice acceptor sites), or branch point sequences within
the neighbouring introns. The earliest evidence for successful exon
skipping of the DMD gene and restoration of dystrophin protein
was obtained in vitro in human lymphoblastoid and mdx mouse
muscle cells (Takeshima et al., 1995; Pramono et al., 1996;
Dunckley et al., 1998). Subsequently, numerous in vitro exon
skipping studies have been undertaken including demonstration
of precise skipping of dmd exon 23 in mdx mouse muscle cells
(Wilton et al., 1999), exon 46 skipping in patient-derived myoblasts (van Deutekom et al., 2001) and more recently the selection
and optimization of human DMD exon 51 skipping antisense
oligonucleotides in preparation for Phase I clinical trials
(Aartsma-Rus et al., 2003; Arechavala-Gomeza et al., 2007).
Convincing pre-clinical evidence for the therapeutic potential of
exon skipping for DMD has come from numerous in vivo studies in
animal models of the disease, most notably the mdx mouse, but also
in humanized DMD mice (Bremmer-Bout et al., 2004; Heemskerk
et al., 2009) and canine models of muscular dystrophy (McClorey
et al., 2006; Yokota et al., 2009). The mdx mouse is a
dystrophin-deficient mutant that arises from the effects of a single
nucleotide base substitution and introduction of a premature termination codon in exon 23 of the DMD gene (Bulfield et al., 1984;
Sicinski et al., 1989). The phenotype of this mouse is relatively mild
with a close to normal lifespan, but it has nevertheless proved an
excellent model in which to demonstrate and fine-tune methods
for molecular correction of the dystrophin defect. Evidence for
in vivo correction of the dystrophin protein defect by antisense
oligonucleotide-mediated exon skipping was first shown in mdx
mice in 2001 (Mann et al., 2001) and this led to subsequent
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experiments in which restoration of dystrophin protein expression
and function was observed (Lu et al., 2003). In these experiments,
Partridge, Wilton and colleagues injected 20 OMe modified antisense
oligonucleotides (RNA analogues incorporating methyl modifications
at the 20 ribose position), the sequences of which were complementary to the mouse DMD exon/intron 23 boundary region, into the
tibialis anterior muscles of adult mdx mice. These authors were able
to detect corrected DMD pre-mRNA transcripts lacking the mutated
exon 23 (i.e. successful exon 23 skipping) and demonstrated restored
dystrophin protein expression with the correct sarcolemmal cellular
localization required for physiological function. Moreover, this
indeed led to restored dystrophin function both in terms of reconstituted molecular expression of dystrophin-glycoprotein complex
protein components (demonstrating that the restored dystrophin
protein contained the requisite functional domains for essential
protein-protein interactions) and improved isometric force generation in treated muscles.
Two limitations to antisense oligonucleotide-mediated splicemodulation were clear at the time of this work in 2003. First,
despite phosphorothioate modification, the 20 OMe antisense
oligonucleotides required co-administration with a co-polymer
(F127) to facilitate cell uptake following intramuscular injection,
suggesting that in vivo delivery of antisense oligonucleotides
would be challenging. Secondly, the level of restored dystrophin
protein was low. These data were confirmed by other groups, and
as a consequence one important area in which subsequent
research effort was focused has been to develop antisense oligonucleotides with improved exon skipping efficiency, principally
through the investigation of new generations of antisense oligonucleotides with enhanced chemical properties. Morpholino
phosphorodiamidate (PMO) antisense oligonucleotides have a
stable, uncharged backbone, in contrast to the negatively
charged 20 OMe phosphorothioate compounds, and were shown
initially to induce mdx DMD exon 23 skipping successfully both
in vitro (Gebski et al., 2003) and in vivo (Fletcher et al., 2006,
2007). A second uncharged antisense oligonucleotide compound,
peptide nucleic acid, was also shown to have good exon skipping
activity in mdx mice (Ivanova et al., 2008a, b; Yin et al., 2008a).
As a result, there now exist several antisense oligonucleotide candidates with distinct backbone chemical properties for clinical
trials. An alternative approach entirely to the use of short synthetic
antisense oligonucleotides has been to encode appropriate exon
skipping antisense sequences within DNA expression vectors,
offering the prospect both of improved delivery (since such
approaches can make use of viral vector delivery methods) and
duration of effect, and also improved skipping efficiency. In the
first in vivo report of this work, Goyenvalle and colleagues (2004)
were able to demonstrate persistent DMD exon 23 skipping in
mdx mice by expression of antisense sequences linked to a U7
snRNA in an adeno-associated virus vector (Goyenvalle et al.,
2004). This approach arose from earlier in vitro work using
U1 snRNA backbones (De Angelis et al., 2002), the natural function of which involves recognition of 50 splice junctions
in pre-mRNAs—hence their powerful utility for such applications,
and which have subsequently shown evidence of good systemic activity and phenotypic improvement in mdx mice (Denti
et al., 2006).
M. J. A. Wood et al.
Phase I clinical trials
demonstrate proof-of-concept
for DMD splice-modulation
Following the initial demonstration of DMD splice-modulation and
functional restoration in mdx mice in 2003 (Lu et al., 2003),
groups both in The Netherlands and the UK have worked towards
clinical evaluation of this splice-correction therapy in patients with
DMD. Several important considerations arise in the translation of
this therapeutic approach to the clinic. First, there is significant
genetic heterogeneity amongst patients with DMD, and therefore
not all DMD mutations will be amenable to dystrophin restoration
via exon skipping, and those that are (the majority) will require
skipping of a range of different exons corresponding to the precise
mutation in order to restore a viable DMD pre-mRNA reading
frame. It has been estimated, according to the Leiden muscular
dystrophy database, that successful single- and multi-exon skipping of all treatable deletions, point mutations and duplications
could provide a disease modifying therapy for 83% of all
patients with DMD (Aartsma-Rus et al., 2009a). Moreover,
while this therapeutic approach is quasi mutation-specific, to the
extent that skipping of specific exons is required to treat particular
groups of mutations, the majority of deletions cluster in ‘hotspot’
regions between DMD exons 43 and 53, which are potentially
correctable by skipping a relatively small number of exons.
Amongst these, the highest frequency was found to be associated
with exon 51, effective skipping of which could treat 13% of all
DMD patients (19% of all patients with gene deletions) and
restore the open reading frame in a cluster of deletions affecting
exons 45–50, 47–50, 48–50, 49–50, 50, 52 and 52–63
(Aartsma-Rus et al., 2003). Secondly, initial clinical trial design
could not permit the testing of healthy volunteers (since exon
skipping in such normal individuals would yield an out-of-frame
DMD transcript), nor could it offer the prospect of therapeutic
efficacy end-points in the first group of treated patients, since
primarily for safety reasons it was decided that such initial trials
would recapitulate almost exactly the 2003 experiment by Lu and
colleagues and involve administration of the antisense oligonucleotide into a single lower limb muscle. Both the Dutch and UK teams
therefore conducted independent Phase I clinical trials directed at
skipping of DMD exon 51, in small numbers of patients in whom
skipping of exon 51 would be predicted to yield an in-frame DMD
transcript and functional dystrophin protein. The Dutch team
(led by the RNA therapy company Prosensa) selected an antisense
oligonucleotide of 20 OMe phosphorothioate chemistry, identical to
that in the 2003 experimental study by Lu et al. (2003) and a
20-mer sequence (PRO051) designed to skip human exon 51 was
injected unilaterally into the tibialis anterior muscles of four
patients with DMD. The results of this trial proved encouraging,
with the treatment being well tolerated and up to 35%
dystrophin-positive fibres detected in biopsy samples from all treated patients (van Deutekom et al., 2007). On the basis of developments in antisense oligonucleotide backbone chemistry
following 2003 (Gebski et al., 2003; Fletcher et al., 2006,
2007), the UK team selected a PMO antisense oligonucleotide
Neuromuscular disease splice therapy
(working in collaboration with AVI Biopharma) for their clinical
trial and conducted an extensive screen of antisense oligonucleotide sequences to maximize exon skipping efficiency, before
selecting the most efficacious 30-mer sequence targeting exon
51 (Arechavala-Gomeza et al., 2007). In this clinical trial, a
single administration of PMO antisense oligonucleotide
(AVI-4658) was injected unilaterally into the extensor digitorum
brevis muscles of seven patients, in a single-blind, dose-escalation
protocol that included a placebo control administered to the contralateral extensor digitorum brevis muscle. This study too was
highly encouraging. PMO antisense oligonucleotides were also
well tolerated by all patients, and at the higher dose received by
five of the seven patients, de novo dystrophin protein expressed at
up to 42% of normal levels in dystrophin-positive fibres (Kinali
et al., 2009). Thus, two clinical studies reporting independently
have highlighted the potential of this splice-correction RNA therapy for DMD.
Current challenges in DMD
splice-modulation
Following the encouraging clinical progress above, further Phase I/
II clinical trials are in progress in both the UK (http://clinicaltrials
.gov/ct2/show/NCT00844 597) and The Netherlands to evaluate
systemic administration in DMD patients of the corresponding
antisense oligonucleotides (PMO and 20 OMePS, respectively)
targeting dystrophin exon 51; the results of both should be
reported in late 2009/early 2010. Results from these clinical
trials will inform future research, but given the encouraging data
already obtained, it is clear that future research must now focus
on the critical scientific and other barriers that currently limit successful clinical application of antisense oligonucleotide
splice-modulation therapy for DMD. In investigations of all novel
RNA-based therapies, including RNA interference-based therapies
as well as antisense oligonucleotide therapies, in vivo delivery of
the therapeutic compounds represents the single greatest obstacle
to successful clinical exploitation. In the case of DMD, there are
two pertinent issues. First, the nature of the disease process itself
involving muscle degeneration leads to disturbed sarcolemmal
integrity and hence a reduced barrier to antisense oligonucleotide
delivery into muscle that works in favour of effective antisense
oligonucleotide delivery in this specific case (Hoffman, 2007).
However, second, given that it is a systemic disease affecting
all peripheral skeletal muscles, including diaphragm, smooth and
cardiac muscle and also the brain, development of a successful
splice-modulation therapy is critically dependent on effective antisense oligonucleotide delivery to and splice-modulation in all
affected tissues. Systemic intravenous delivery of both 20 OMe
and PMO antisense oligonucleotides has been shown to restore
dystrophin protein expression in the dystrophin-deficient mdx mice
(Lu et al., 2005; Alter et al., 2006). However, in these experiments, high dose (up to 100 mg/kg), multi-injection antisense
oligonucleotide administration was required to generate detectable
dystrophin protein expression in multiple peripheral muscle groups.
Even at such doses the correction efficiency remained relatively
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low for both antisense oligonucleotide chemistries, up to 16 and
25% dystrophin-positive fibres with 20 OMe and PMO antisense
oligonucleotides, respectively, following three injections (Lu et al.,
2005; Alter et al., 2006), with focal and highly variable levels
of protein restored in different muscle groups. These findings
suggested that at best antisense oligonucleotide splice-correction
therapy may have only partial clinical efficacy in DMD patients,
unless higher equivalent doses can be used in human subjects or
improved antisense oligonucleotide delivery and/or administration
regimens are developed. A second related scientific barrier to successful clinical application is that in neither of these high dose
antisense oligonucleotide studies was dystrophin protein shown
to be restored in mdx heart muscle. This is an important consideration given that cardiac problems are found in nearly all (up to
90%) patients with DMD patients, a significant number of whom
develop a dilated form of cardiomyopathy leading to cardiac death
(Bushby et al., 2003). Moreover, there are concerns that a method
for the effective correction of the skeletal muscle dystrophin
defect, in the absence of cardiac correction, might worsen cardiac
disease progression in DMD (Townsend et al., 2008). Thus the
development in parallel of methods to correct the cardiac DMD
phenotype is of great importance.
Novel methods for improved systemic antisense oligonucleotide
delivery have thus been intensively investigated with the aim
of permitting lower antisense oligonucleotide doses to be used,
compatible with acceptable safety and toxicity profiles, and to
facilitate more effective systemic antisense oligonucleotide delivery
to all DMD-affected tissues, ideally targeted specifically to such
tissues. Recently a number of groups have independently reported
that the systemic intravenous delivery of uncharged PMO antisense oligonucleotides can be enhanced significantly by direct
PMO conjugation to short, arginine-rich, cell-penetrating peptides
(Jearawiriyapaisarn et al., 2008; Wu et al., 2008, 2009; Yin et al.,
2008b). Cell-penetrating peptides are short peptides (mostly cationic) that were found to impart cell-penetrating properties
to attached cargoes. They were initially identified as sections of
proteins (protein transduction domains), for example the argininerich nuclear localization signal of the HIV-1 trans-activator of transcription protein Tat (Vives et al., 1997) and the well-described
helix 3 peptide of the Drosophila ‘Antennapedia’ gene transcriptional activator (which became known as Penetratin) (Derossi
et al., 1994). Later, synthetic variants of such cell-penetrating
peptides were developed, of which the most effective in the
case of PMO and peptide nucleic acid were a peptide family the
parent of which is known as ‘R’, a 14-mer that contains four
contiguous Arg-Aminohexanoyl-Arg (R-X-R) motifs (Nelson
et al., 2005). Pharmacologically, R-PMO has shown a 2-fold
increase in elimination half-life, 4-fold increase in volume of distribution, and better tissue uptake when injected into rats compared to unconjugated PMO (Nelson et al., 2005). More recently
another family of arginine-rich peptide derivatives based on
Penetratin extended by six Arg residues (R6Pen) (Abes et al.,
2007) and their serum-stabilized versions (Pip) have been
advanced (Ivanova et al., 2008a, b). Commercially, the leading
peptide at present is known as ‘B’: a 14-mer peptide variant of
‘R’ with sequence (R-X-R-R-B-R)2-X-B, where ‘B’ is b-Alanyl,
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which is thought to have a favourable toxicology profile (Stein
et al., 2008).
In all of the R-PMO and B-PMO studies to date, highly
significant body-wide dystrophin protein restoration in multiple
peripheral muscle groups at low PMO doses has been reported,
with some limited correction in cardiac muscle seen for the first
time, and with convincing amelioration of the mdx mouse dystrophic phenotype (Jearawiriyapaisarn et al., 2008; Wu et al., 2008,
2009; Yin et al., 2008b). These studies have therefore highlighted
the potential of peptide-PMO antisense oligonucleotide conjugates
as novel and powerful therapeutic agents for DMD. B-PMO (also
known simply as PPMO) is the lead PMO candidate compound
for DMD being developed by AVI Biopharma, the first clinical trial
of which is likely to take place in the USA targeting DMD exon
50. However, these early studies have yet to explore the full range
of peptide design space, and in particular have not yet investigated methods for specific peptide targeting of muscle and heart
and other affected tissues. Recently, the first report of such
muscle-targeted antisense oligonucleotide delivery demonstrated
that chimeric peptides incorporating muscle-specific heptapeptide
and cell-penetrating peptide domains (prototype B-MSP-PMO)
could indeed yield targeted antisense oligonucleotide delivery and
improved dystrophin splice correction in mdx mice (Yin et al.,
2009). Thus PPMO drugs have considerable potential as therapeutic agents but given their novelty, extensive safety testing of these
compounds will be required before first-in-man studies can
proceed. Finally, while at an early stage, recent developments in
nanotechnology look promising for providing antisense oligonucleotide delivery for DMD. In one study, nanoparticles were
successfully engineered to deliver antisense oligonucleotides for
exon skipping via intramuscular injection in mdx mice (Sirsi
et al., 2009) and in another, cationic nanoparticles incorporating
a low dose of 20 OMePS antisense oligonucleotides were delivered
via the intraperitoneal route to mdx mice and showed evidence of
systemic activity and low level dystrophin restoration (Rimessi
et al., 2009).
Dystrophin correction in heart
and other affected tissues
As successful methods are developed to enhance the systemic
delivery and activity of antisense oligonucleotides for DMD
therapy, the question of efficacy in affected non-muscle tissues,
especially heart, will become increasingly important. DMD is a
systemic disease, and cardiomyopathy is the second leading
cause for the death in patients with DMD, accounting for
10–40% of fatalities in DMD populations (Cox and Kunkel,
1997; Muntoni et al., 2003). Even female carriers are reported
to have a higher risk of developing cardiomyopathy (Politano
et al., 1996; Saito et al., 1996). There is increasing emphasis on
early cardiac intervention in DMD patients with medical treatments including angiotensin-converting enzyme inhibitors alone,
or in combination with beta-blockers and glucocorticosteroid therapy (Markham et al., 2008; Ogata et al., 2009). However, there
are currently a lack of good efficacy data for long-term use of
M. J. A. Wood et al.
these treatments in patients with DMD (Finsterer, 2009; Ogata
et al., 2009). Ultimately there is a clear need for new molecular
therapies to correct the cardiac dystrophin defect as well. In the
case of exon skipping, the degree to which cardiac correction can
be achieved and disease progression delayed will depend on the
efficiency of antisense oligonucleotide delivery to the heart, the
degree of dystrophin splice-modulation achieved, and crucially, to
what extent the dystrophin isoform successfully generated has
functional utility in heart. In this regard it should be noted that
there is wide heterogeneity in the BMD cardiac phenotype, but
that in general such patients typically have a higher incidence of
cardiac disease although often this is subclinical and late onset
(Finsterer and Stollberger, 2003).
Despite very high antisense oligonucleotide doses, systemically
administered 20 OMe and PMO antisense oligonucleotides failed to
demonstrate evidence of splice-modulation in mdx mouse heart
(Lu et al., 2005; Alter et al., 2006). A more recent study, utilizing
even higher doses (nine doses at 100 mg/kg), reported very low
levels (1–2%) of dystrophin splice-modulation in heart with
both antisense oligonucleotide chemistries (Heemskerk et al.,
2009). The reasons for the low efficiency cardiac dystrophin
splice-modulation are unclear, but are probably related to the
poor ability of unmodified antisense oligonucleotides to traverse
the endothelial barrier in heart microvasculature and/or enter cardiomyocytes with sufficient efficacy to yield detectable dystrophin
correction. However, more recent work from several laboratories
with PMO compounds has shown evidence of significantly
restored cardiac dystrophin protein for the first time, albeit at
low levels compared with skeletal muscle (Jearawiriyapaisarn
et al., 2008; Wu et al., 2008, 2009; Yin et al., 2008b). This is
encouraging and important since it demonstrates that there is no
intrinsic barrier within cardiomyocytes preventing effective dystrophin exon skipping and suggests that a solution to cardiac
antisense oligonucleotide delivery can be found. Nevertheless,
the precise function of cardiac dystrophin and the level of restored
dystrophin that would be required for cardiac correction and
disease modification in DMD patients remain unclear. There are
fundamental structural differences between cardiac and skeletal
muscles with respect to dystrophin, e.g. dystrophin expression pattern and the localization of dystrophin within T-tubules in cardiac
but not skeletal muscle (Porter et al., 1992; Klietsch et al., 1993;
Kaprielian et al., 2000; Rybakova et al., 2000; Yasuda et al.,
2005; Duan, 2006), all of which might contribute to different
functional requirements for dystrophin in heart and to variation
in the efficacy of antisense oligonucleotide activity in these two
types of muscle. Recently, in unpublished work, we have demonstrated the striking potential of Pip series cell-penetrating peptides
(Ivanova et al., 2008a, b) to restore cardiac dystrophin protein
when attached to PMO. In particular, the activity of a new peptide Pip5e has resulted in almost complete cardiac dystrophin
restoration in mdx mice following intravenous delivery (Wood,
Gait and Yin, unpublished data). This was unexpected and at present the mechanism is not known since the central ILFQY domain
with Pip5e is not known to be a heart-specific sequence.
Nevertheless such data point towards future research needed to
optimize antisense oligonucleotide therapy for DMD fully. Such
discoveries are also likely to be of direct relevance to the
Neuromuscular disease splice therapy
development of biological therapies for other neuromuscular and
cardiac disorders which face similar delivery challenges.
Brain and retina are two other major non-muscle sites of
dystrophin expression. Full-length dystrophin (Dp427) and other
isoforms (Dp140 and Dp71) are expressed in cerebral cortex, cerebellum, hippocampus and Purkinje cells (Lidov et al., 1990).
About 30% of DMD patients show non-progressive intellectual
and/or cognitive impairment (Bresolin et al., 1994; Wicksell
et al., 2004) and a sub-set show an increased incidence of psychiatric disorders; e.g. autism, dysthymic and depressive disorders
(Komoto et al., 1984; Fitzpatrick et al., 1986; Ogata et al., 2009).
Interestingly, a recent study has suggested that dystrophin loss
influences emotionally defensive behaviour in mdx mice and that
this behavioural change can be ameliorated by the restoration of
dystrophin induced by direct intracerebroventricular PMO antisense oligonucleotide treatment (Sekiguchi et al., 2009).
However the role of dystrophin in brain is not fully understood.
In retina, three different dystrophin isoforms exist; Dp427, Dp71
and Dp260—the latter being the dominant form. Dp427 is mainly
expressed at the photoreceptor terminal and its deficiency generates abnormal neurotransmission. Most DMD patients show
abnormalities in retinal electrophysiology (reduced b-wave in
electroretinography), indicating disturbed neurotransmission
(Cibis et al., 1993; Fitzgerald et al., 1994), and a high proportion
also show defects in colour vision (Costa et al., 2007). Thus, ultimately, appropriate methods will need to be developed to target
the delivery of antisense oligonucleotides to all DMD-affected tissues including brain and retina in order to correct fully all elements
of the DMD phenotype.
Multi-exon skipping in the
dystrophin gene
A significant limitation of the current splice-modulation methods
for DMD is while antisense oligonucleotide compounds are not
‘personalized’ as such, each antisense oligonucleotide is developed
for skipping of a specific exon (e.g. exon 51) and therefore for
treating a specific sub-set of mutations and DMD patients. As a
result, several antisense oligonucleotides will need to be developed
and evaluated in clinical studies in order to treat a sizeable proportion of all patients with DMD—estimates suggest that the top
10 antisense oligonucleotides could treat in the order of 40% of
all patients (Aartsma-Rus et al., 2009a). Thus, regulatory agencies
may need to re-evaluate their approach to the development and
testing of personalized genetic medicines, such as antisense oligonucleotides for DMD, in order to permit the fast-tracking of
‘classes’ of antisense oligonucleotide compounds (rather than specific antisense oligonucleotide sequences) for clinical approval.
However, an alternative scientific approach would be to reduce
the overall number of antisense oligonucleotides required by engineering them to skip multiple, non-adjacent DMD exons, thereby
providing a more ‘universal’ antisense oligonucleotide for DMD
treatment. Studies to date that have investigated multi-exon skipping (Aartsma-Rus et al., 2006b; Fall et al., 2006; Aartsma-Rus
and van Ommen, 2007; van Vliet et al., 2008) have largely
Brain 2010: 133; 957–972
| 965
evaluated combinations or cocktails of individual antisense oligonucleotides, each targeting separate DMD exons, in attempts to
generate a more widely applicable therapeutic combination.
However such studies illustrate the challenge of modulating alternative splicing within a complex pre-mRNA transcript such as the
DMD pre-mRNA. The DMD transcript comprises 79 exons stretching over 2.2 Mb on the X chromosome, transcriptional processing
of which results in the splicing of upstream exons (e.g. exon 43)
hours before downstream exons (e.g. exon 53) are processed
(Tennyson et al., 1995). It will therefore be important to acquire
a more detailed knowledge of alternative splicing in the DMD
gene and how this is regulated, particularly in the region from
exons 40-55 where a high proportion of DMD deletions are clustered. Such fundamental splicing knowledge is now starting to be
obtained. For example the likelihood of a given exon being
included in the mature mRNA transcript is known to be dependent
on the rate of gene transcription, and the relative size of neighbouring introns and exons (Matlin et al., 2005). What will be
required in the future will be the development of methods
based on single antisense oligonucleotides that are able to perturb alternative splice regulation simultaneously at multiple
loci within a given pre-mRNA transcript. This will not only open
the door to generic ‘universal’ splice-modulating antisense
oligonucleotides for all patients with DMD but will probably provide tools that allow more intelligent modulation of gene activity
in general.
Antisense oligonucleotide
splice-correction for other
neuromuscular disorders
The clinical progress in evaluating antisense oligonucleotide
splice-modulation therapy for DMD and the understanding of
how to deliver and target such antisense oligonucleotides systemically to affected tissues opens the possibility of this method being
exploited more widely to treat the range of neuromuscular
diseases related to splicing. A number of neuromuscular diseases
with a link to defects alternative splicing are shown in Table 1.
After DMD, perhaps those that appear the most tractable targets
for antisense oligonucleotide therapy are SMA and myotonic
dystrophy.
SMA is a group of mostly inherited disorders that cause motoneuron degeneration and constitute a leading genetic cause of
infant mortality (Talbot and Davies, 2001). Autosomal recessive
SMA is the commonest form and arises due to loss-of-function
mutations in the SMN1 (survival motoneuron) gene. As the
nomenclature suggests, an SMN2 gene exists which encodes the
same open reading frame, and which is thought to be an SMN1
paralogue that arose by evolutionary duplication. However, SMN2
incorporates a C4T single nucleotide substitution in exon 7 that,
without altering the amino acid encoded, alters SMN2 pre-mRNA
splicing probably via the elimination of an exonic splicing enhancer
containing an alternative splicing factor/splicing factor 2 regulatory
factor binding site (Cartegni and Krainer, 2003) and/or the creation of an exonic splicing silencer associated with the splicing
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| Brain 2010: 133; 957–972
repressor heterogeneous nuclear ribonucleoprotein A1 (Kashima
and Manley, 2003). This results in the generation of less than
10% of normal functional SMN protein from this locus (Burghes
and Beattie, 2009). As a consequence there is considerable heterogeneity in the SMA phenotype, with severe SMA occurring when
levels of functional SMN protein are reduced by 80% (Lefebvre
et al., 1997). However, SMN levels can be naturally modulated by
the number of genomic SMN2 copies in individual patients,
thereby modifying the disease phenotype. SMN has a ubiquitous
and essential function involving correct assembly of small nuclear
ribonucleoprotein/spliceosomal complexes required for pre-mRNA
splicing. However, SMN’s neuronal- or motoneuronal-specific
function is largely unknown and therefore the precise mechanism
of motoneuron degeneration remains elusive. Nevertheless, recent
M. J. A. Wood et al.
evidence suggesting tissue-specific splicing dysregulation, perhaps
due to the fact that the small nuclear ribonucleoprotein content in
different tissues is sensitive to differing degrees of SMN loss
(Zhang et al., 2008), may provide new insight (Talbot and
Davies, 2008; Zhang et al., 2008; Burghes and Beattie, 2009).
A potential therapeutic approach for SMA would therefore be to
increase expression of functional SMN transcripts from the intact
SMN2 loci, which are largely non-functional due to exon 7 exclusion in pre-mRNA processing. The challenge to develop a
splice-correction therapy for SMA would therefore involve intervention to redirect splicing of exon 7 of the SMN2 pre-mRNA to
favour inclusion rather than exclusion of the exon (Fig. 3), and
therefore increase the level of functional SMN protein. This is
however more challenging than conventional exon skipping
Figure 3 Antisense oligonucleotide-mediation splice-correction for other neuromuscular diseases. In DMD patients with an exon 50
deletion, exon 51 can be excluded by targeting the exon 51 region, including at the exonic splicer enhancer (ESE) site, with short
homologous antisense oligonucleotide sequences to restore the DMD reading-frame and produce functional dystrophin protein (A). In
other neuromuscular diseases, typified by SMA, therapeutic effects will require not exon exclusion but inclusion. SMA results from SMN1
loss-of-function, however an SMA modifier gene SMN2 exists incorporating a single nucleotide modification in exon 7 that abolishes an
exonic splicing enhancer leading to low level production of functional SMN protein from this locus. Strategies to enhance exon 7 inclusion
within the SMN2 pre-mRNA are therefore predicted to yield enhanced functional SMN protein synthesis. An example of such an approach
developed by Hua et al. (2008) is shown in B, whereby antisense oligonucleotides directed to inhibit the intronic splice silencer site (ISS) in
intron 7 of the SMN2 gene have a powerful effect on pre-mRNA processing, leading to a greater probability of exon 7 inclusion within the
mature mRNA transcript and resulting in the increased production of normal SMN protein (Hua et al., 2008). Myotonic dystrophy (DM1)
is caused by RNA gain-of-function due to an expanded CUG repeat in the DMPK transcript. Possible therapeutic methods are shown in C
and include; antisense oligonucleotide- or siRNA-mediated knockdown of the CUG repeat RNA and inhibition of the RNA gain-of-function
with antisense oligonucleotides or small molecule drugs that release sequestered MBNL1 protein and correct the dysregulated splicing
phenotype.
Neuromuscular disease splice therapy
(as described above for DMD), since instead of blocking splicing
(to exclude an exon) a positive effect must be generated to suppress the signals that are present to direct natural exon exclusion.
Approaches to enhance exon 7 inclusion have included screens for
small molecules (Zhang et al., 2001), trans-splicing methods
(Coady et al., 2007) as well as antisense oligonucleotide-based
strategies. Proof-of-principle for the use of antisense oligonucleotides was initially obtained in cell culture to block the 30 splice site
of exon 8 (Lim and Hertel, 2001). These methods have been
further developed using bifunctional antisense oligonucleotides
that directly target exon 7 and which incorporate exonic splicing
enhancer motifs to enhance exon recognition, recruit Serine/
aRginine (SR) proteins and lead to an increased probability of
exon inclusion (Skordis et al., 2003). Similarly the design of bifunctional antisense oligonucleotides covalently coupled to aRginine/
Serine (RS) peptides (i.e. a sequence corresponding to a functional
domain in the SR protein itself) that activate splicing and exon 7
inclusion through the recruitment of spliceosomal components
has also been successful (Cartegni and Krainer, 2003). While
such methods can yield splice-correction and exon 7 inclusion,
the efficiency of this process is low, and much subsequent work
has focused on attempts to improve antisense oligonucleotide efficiency and to discover more favourable SMN2 target sequences.
In contrast to the use of long bifunctional antisense oligonucleotides, a recent study exploited a very short antisense oligonucleotide (an 8-mer) that was identified to target a GC-rich octamer
region (CUGCCAGC) within intron 7 that partially overlaps with a
known intronic splicing silencer motif. This antisense oligonucleotide gave powerful splicing redirection and exon 7 inclusion at low
nanomolar concentrations in fibroblasts of patients with SMA,
with high specificity and in the absence of detectable off-target
effects (Singh et al., 2009). In a distinct approach, Krainer and
colleagues (Hua et al., 2007) undertook a wide screen using a
different antisense oligonucleotide chemical backbone containing
20 -methoxyethyl ribose base modifications, arrayed across exon 7,
and identified several novel 20 -methoxyethyl riboses that promoted efficient exon 7 inclusion and also, interestingly, that did
not correspond to previously well-characterized regulatory motifs
(Hua et al., 2007). Further analysis revealed that some of the most
effective of these antisense oligonucleotides mapped to putative
intronic splicing silencer sequences within intron 7, and in particular to heterogenous ribonucleoprotein A1/A2 motifs (heterogenous ribonucleoprotein A/B family proteins that affect both 50
and 30 splice-site selection), providing a mechanistic explanation
for their enhanced activity. Further high resolution antisense oligonucleotide screens in this intronic splicing silencer region yielded
two new antisense oligonucleotides that showed highly effective
exon 7 inclusion (up to 90%) in vitro and remarkably also in vivo
(Hua et al., 2008), principally in the liver and kidney following
intravenous administration in human SMN2 transgenic mice, but
with little evidence of splice correction in spinal cord.
Ultimately, for a successful SMN splice-correction therapy,
potent antisense oligonucleotides that promote highly effective
exon 7 inclusion will need to be coupled with methods
that allow antisense oligonucleotide delivery to target motoneurons within the CNS. The in vivo activity of such antisense
oligonucleotides within the CNS has been recently shown via
Brain 2010: 133; 957–972
| 967
intracerebroventricular delivery (Baughan et al., 2009; Williams
et al., 2009); Williams and colleagues (2009) demonstrating
phenotypic improvement in SMN7 mice in their study and confirming the therapeutic potential of such antisense oligonucleotides
once delivered at high concentration to the appropriate CNS compartment. While systemic delivery of SMN splice-correcting
antisense oligonucleotides to the CNS remains a major goal, this
has yet to be convincingly demonstrated, although low efficiency
SMN expression has been recently shown to be restored in parts
of the CNS in a single study (Dickson et al., 2008). An alternative
delivery approach for the correction of SMN2 pre-mRNA splicing,
as discussed earlier for DMD, is the utilization of viral expression
methods that incorporate U7 snRNA backbones for the targeted
expression of antisense sequences (Madocsai et al., 2005; Marquis
et al., 2007). Using the U7 methodology to express bifunctional
antisense sequences that promote intron 7 inclusion has now been
shown to rescue the most severe SMA mouse model, when delivered by germline transgenesis (Meyer et al., 2009), testifying to
their ability to significantly modify disease progression if delivered
appropriately and at an early stage in the disease course.
Therefore such U7-based methods, coupled with highly efficient
viral delivery using adeno-associated virus-9 e.g. that permit delivery across the blood-brain barrier (Foust et al., 2009), are likely to
have therapeutic potential.
A second neuromuscular disease linked to aberrant splicing and
that has been shown in principle to be susceptible to directed
antisense correction is myotonic dystrophy, both type 1 (DM1)
and type 2 (DM2) (Ranum and Cooper, 2006). In DM1, the presence of expanded CTG repeats within the 30 untranslated region
of the DMPK gene leads to transcription of an RNA containing an
expanded CUG repeat beyond the normal range of 5–38 repeats,
resulting in an RNA gain-of-function that causes neuromuscular
disease and widespread dysregulation of alternative splicing leading to multi-system dysfunction. In DM2 a CCTG expansion in the
zinc finger protein 9 (ZNF9) gene leads to splicing misregulation
and disease. The underlying disease mechanism appears to be that
the expanded RNA repeat region forms a hairpin containing C-G
base pairs that sequesters and disrupts the functions of specific
RNA binding proteins (Philips et al., 1998). These include RNA
binding proteins with particular affinities for CUG RNA repeats
such as CUG binding protein 1 and muscleblind-like 1 family proteins (O’Rourke and Swanson, 2009), which regulate RNA
processing and alternative splicing. A second component
of DM1 pathogenesis appears to involve aberrant activation of
protein kinase C which leads to up-regulated CUG binding protein 1 expression (Kuyumcu-Martinez et al., 2007). Together
muscleblind-like 1 and CUG binding protein 1 tightly regulate
the transition between the expression of embryonic and adult
splice variants that occurs in post-natal skeletal muscle, and
which is consequently disrupted in DM1 leading to disease (Lin
et al., 2006).
In such RNA gain-of-function disorders the CUG expanded RNA
represents a viable therapeutic target, inhibition of which is likely
to lead to at least partial disease modification as suggested by data
from an inducible DM1 mouse model (Mahadevan et al., 2006).
As is the case with SMA, inhibitory approaches have included
screens for small molecule inhibitors to block the binding of
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| Brain 2010: 133; 957–972
muscleblind-like 1 to the CUG expanded RNA repeat in DM1 (or
the CCUG expanded repeat in DM2), as well as antisense
oligonucleotide-based methods. Very recently Warf et al. (2009)
undertook a limited small molecule library screen of compounds
with affinity for structured nucleic acid and identified a compound
pentamidine that was shown to block muscleblind-like 1 binding
to the repeat expanded RNA. They obtained encouraging data
both in vitro and in vivo in a DM1 mouse model expressing
250 repeats within the 30 untranslated region of the human
skeletal -actin gene, and which led to partial reversal of the
misregulated splicing phenotype, suggestive of pentamidine efficacy (Warf et al., 2009). To what extent such compounds can
reverse all pathogenic events in DM1 or are without off-target
effects on other structured RNA elements remain to be
established. A similar approach using antisense oligonucleotidemediated inhibition of the expanded RNA has also recently
been reported (Wheeler et al., 2009). Wheeler and colleagues
utilized a 25-mer PMO antisense oligonucleotide containing multiple complementary CAG triplet repeats designed to bind the
CUG repeat region of the expanded RNA with high affinity.
They were able to demonstrate inhibition of muscleblindlike 1 binding to the expanded RNA and redistribution of
muscleblind-like 1 protein from nuclear RNA foci with most
likely subsequent degradation of the RNA. With release of
muscleblind-like 1 from the RNA foci, the defect in alternative
splicing regulation was found to be at least partially corrected,
resulting in restored ion channel function following intramuscular
injection in HSALR transgenic mice that show similar alternative
splicing defects to those found in DM1 (Wheeler et al., 2009).
In another recent related but distinct antisense oligonucleotide
approach, 20 OMePS antisense oligonucleotides were used, not to
modify splicing or to interrupt interaction with muscleblind-like 1,
but to suppress the expression of the expanded DMPK RNA
directly (Mulders et al., 2009). This CAG repeat-containing antisense oligonucleotide was found to suppress DMPK RNA expression by 90%, reduce RNA aggregation in DM1 patient cells and
led to partial reversal of the RNA splicing defect. To what extent
such findings are sufficiently specific to the mutant transcript and
without detrimental off-target effects will need to be rigorously
determined, however such approaches have clear clinical applicability and potential for correcting the disease phenotype in
patients.
Conclusions and future
prospects
Within the last few years the prospect of successful antisense
oligonucleotide splice-modulation therapy for neuromuscular disease has moved a step closer, particularly for DMD but also for
SMA and myotonic dystrophy. The data from the first clinical trials
in DMD patients have proved highly encouraging, and technical
advances in the development of methods for both directed exon
exclusion and inclusion suggest that such antisense oligonucleotide
splice-correction therapies may have broad application. Moreover,
the recent preclinical work in DMD animal models using peptide
M. J. A. Wood et al.
enhanced antisense oligonucleotide delivery, suggests that solutions to the challenge of systemic and tissue-targeted delivery
that will be required for effective treatment are close at hand.
Close cooperation in future with regulatory agencies both in
Europe and the USA will hopefully lead to new ways of
looking at and regulating such ‘personalized’ therapies to permit
their safe and cost-effective testing and the rapid development
of this new generation of medicines that is much needed by
patients.
Acknowledgements
The authors wish to thank Yiqi Seow for expert help in the design
of the figures.
Funding
The UK Medical Research Council, the UK Muscular Dystrophy
Campaign, Action Duchenne and Muscular Dystrophy Ireland
(to M.J.A.W.).
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