NUCLEIC ACID THERAPEUTICS Volume 24, Number 1, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/nat.2013.0451

Skipping Multiple Exons of Dystrophin Transcripts Using Cocktail Antisense Oligonucleotides Yusuke Echigoya1 and Toshifumi Yokota1,2

Duchenne muscular dystrophy (DMD) is one of the most common and lethal genetic disorders, with 20,000 children per year born with DMD globally. DMD is caused by mutations in the dystrophin (DMD) gene. Antisense-mediated exon skipping therapy is a promising therapeutic approach that uses short DNA-like molecules called antisense oligonucleotides (AOs) to skip over/splice out the mutated part of the gene to produce a shortened but functional dystrophin protein. One major challenge has been its limited applicability. Multiple exon skipping has recently emerged as a potential solution. Indeed, many DMD patients need exon skipping of multiple exons in order to restore the reading frame, depending on how many base pairs the mutated exon(s) and adjacent exons have. Theoretically, multiple exon skipping could be used to treat approximately 90%, 80%, and 98% of DMD patients with deletion, duplication, and nonsense mutations, respectively. In addition, multiple exon skipping could be used to select deletions that optimize the functionality of the truncated dystrophin protein. The proof of concept of systemic multiple exon skipping using a cocktail of AOs has been demonstrated in dystrophic dog and mouse models. Remaining challenges include the insufficient efficacy of systemic treatment, especially for therapies that target the heart, and limited long-term safety data. Here we review recent preclinical developments in AO-mediated multiple exon skipping and discuss the remaining challenges.

Sarepta Therapeutics and Prosensa/GlaxoSmithKline (GSK) are currently conducting phase 2b and 3 trials for exon 51 skipping using two different types of AOs, phosphorodiamidate morpholino oligomers (PMOs) and 2¢O-methylated phosphorothioates (2’OMePSs), respectively (ClinicalTrials.gov identifiers NCT01396239 and NCT01254019) (Cirak et al., 2011, 2012; Goemans et al., 2011). Prosensa/GSK is also conducting a phase 1/2a trial with 2’OMePS AOs that target exon 44 (ClinicalTrials.gov identifier: NCT01037309). Finally, a phase 1 clinical trial of PMOs targeting exon 53 has started in 2013 in Japan (UMIN-CTR Clinical Trial number UMIN000010964). Although exon skipping is currently a most promising therapeutic strategy for inducing expression of the dystrophin protein in DMD patients, one major challenge is its limited applicability. Notably, AO therapy is a personalized single-exon skipping therapy that is mutation specific (i.e., different AOs are required for skipping of different exons to restore the reading frame). Indeed, many DMD patients need more than a single exon to be skipped. For example, a splice site mutation in intron 6 in dystrophic dogs requires both exon 6 and exon 8 to be skipped (Fig. 1).

Introduction

D

uchenne muscular dystrophy (DMD), the most common fatal genetic disorder, is inherited in an Xlinked recessive fashion, affecting approximately 1 in 3,500 newborn boys regardless of ethnic origin (Zellweger and Antonik, 1975). DMD is a progressive and lethal degenerative disorder, and the mean age of death of DMD patients is approximately 25 years (Eagle et al., 2002). Currently there is no effective treatment for DMD. DMD is caused by the absence of the dystrophin protein due to mutations in the dystrophin (DMD) gene (Duchenne, 1867; Koenig et al., 1987). DMD is one of the longer human genes, with as many as 79 exons (Koenig et al., 1987), and encodes dystrophin, a muscle membrane-supporting protein that connects the muscle fiber cytoskeleton through the cell membrane to the extracellular matrix (Hoffman et al., 1987). Antisense oligonucleotide (AO)-mediated exon skipping, which eliminates exon(s) and restores a functional open reading frame, appears to be one of the most promising novel treatments for DMD and the one that is closest to clinical application (Lee and Yokota, 2013).

1

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada. The Friends of Garrett Cumming Research & Muscular Dystrophy Canada, HM Toupin Neurological Science Research Chair, University of Alberta, Edmonton, Alberta, Canada. 2

57

58

Multiple exon skipping has emerged as an alternative molecular therapeutic modality that may expand the therapeutic applicability of this technique. In this variation, multiple exons are skipped in order to restore the dystrophin mRNA open reading frame. Theoretically, multiple exon skipping could rescue approximately 90% of patients with deletion mutations, 80% of DMD patients that have duplication mutations, and 98% of those with nonsense mutations (Yokota et al., 2007a; Aartsma-Rus et al., 2009; Yokota et al., 2012a). Encouragingly, several studies have shown that using combinations of AOs allows multiple exon skipping. This has been demonstrated in preclinical trials in dystrophic dog and mouse models in vivo and in cell lines from DMD patients and from the dystrophic dog model in vitro (Table 1) (Aartsma-Rus et al., 2004a, 2006a, 2007; McClorey et al., 2006a; Aoki et al., 2012). However, the efficacy of systemic multiple exon skipping therapy must be improved for it to be clinically useful. Here we review recent preclinical tests of AO-mediated multiple exon skipping in animal models of DMD and in DMD patient cell lines and discuss the challenges of multiple exon skipping therapy. The Dystrophin Gene in DMD: Structure and Mutations

Currently, treatment for DMD is limited to palliative care, and DMD patients’ use of daily glucocorticoids is associated with significant side effects. Chronically progressive muscle degeneration results in delayed motor development, including difficulty walking in early childhood, and skeletal muscle weakness, especially in the pelvic girdle, neck flexor, proximal limb, and shoulder girdle muscles. Most DMD patients are wheelchair-bound by the time they are 12–13 years old. Breathing difficulties start by the time they are 20 years old, and patients require ventilation support to manage respiratory insufficiency. DMD patients usually die when they are 20–30 years old due to respiratory or cardiac failure (Finsterer and Stollberger, 2003; Passamano et al., 2012). The DMD gene is the largest known human gene, spanning more than 2.4 million base pairs on the X chromosome. Its full-length 14-kb transcript has 79 exons and is predominantly expressed in skeletal and cardiac muscles (Hoffman et al., 1987; Koenig et al., 1987). The main muscle isoform of the DMD gene has 3,685 amino acids (427 kDa) and plays a central role in organizing a multi-protein complex at the sarcolemma that links cytoskeleton proteins to extracellular matrix proteins and that protects the membrane from damage

FIG. 1. A point mutation at an acceptor splice site (ASS) in the dystrophic dog model and antisense oligonucleotide (AO) design for multiple exon skipping. To rescue the splice site mutation in a dystrophic dog model, at least two exons, exon 6 and exon 8, must be removed from premature mRNA molecules using AOs. Exon 9 can be alternatively spliced. DMD, Duchenne muscular dystrophy.

ECHIGOYA AND YOKOTA

during muscle contraction (Rybakova et al., 2000; Watkins et al., 2000). The full-length dystrophin protein has four domains, namely the actin-binding NH2- (N) terminal domain, the rod domain, the cysteine-rich domain, and the COOH- (C) terminal domain. Most of the protein’s functions are assigned to the N- and C-terminals and to the cysteinerich domain (Bies et al., 1992). In contrast, the central rod domain, which consists of 24 spectrin repeats with 4 hinges and which spans about half the length of the protein, appears less essential for dystrophin function. However, most DMD mutations occur within the rod region, and these mutations interfere with the translation of the functionally important C-terminal domain (Yokota et al., 2009b). Mutations in the DMD gene can be caused by inheritance of the affected allele from a mother who is a DMD carrier, but owing to the large size and the complexity of the gene, about one-third of cases are due to spontaneous new mutations (Caskey et al., 1980; Barbujani et al., 1990). DMD is caused by various mutations of the DMD gene, including deletions, duplications, nonsense mutations, point mutations, and complex rearrangement mutations. However, deletion mutations are the main cause of DMD and Becker muscular dystrophy (BMD), which is caused by a reduction in functional dystrophin. Deletions account for more than half of the mutations (*65%), followed by nonsense (*15%) and duplication mutations (*13%) (Takeshima et al., 2010; White et al., 2006; Magri et al., 2011; Yokota et al., 2012a). In addition to exonic mutations, 7% of DMD/BMD patients harbor intronic mutations (Dent et al., 2005). DMD-causing mutations are mainly out-of-frame ones that disrupt the open reading frame (Aartsma-Rus et al., 2006b). Interestingly, in spite of the mutations, the open reading frame may be maintained in the messenger RNA (mRNA) (termed ‘‘inframe’’ mutations), allowing translation of a truncated, yet partially functional, dystrophin protein. In contrast to DMD, BMD is generally caused by in-frame deletion mutations or duplications (Aartsma-Rus et al., 2006b). Approximately 70% of the dystrophin deletion mutations cluster in the region between exons 45 and 55, which is termed the ‘‘hotspot’’ region (Yokota et al., 2007b; Aartsma-Rus et al., 2009; Magri et al., 2011). These BMDcausing truncated dystrophin proteins differ structurally in different patients due to their distinct mutation patterns. Dystrophin can retain significant functionality even when it is missing large parts (England et al., 1990; Harper et al., 2002; Roberts et al., 2002; Sakamoto et al., 2002; Shin et al., 2012).

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Mutation

Target exons for skipping

Ex 6 & 8

3 PMOs

A nonsense mutation of ex 23

A single G duplication in ex 51 2 bp deletion in ex 20

mdx mice

DMD patients

mdx4cv mice A nonsense mutation of ex 53

3

9 3

9

4

4

Ex 52 & 53

Bodywide muscles by i.m.

Primary myotubes by in vitro transfection Primary myotubes by in vitro transfection Immortalized myotubes by in vitro transfection Tibialis anterior muscle by i.m.

Bodywide muscles by i.v.

Tibialis anterior or forearm muscles by i.m. Tibialis anterior muscle by i.m. Converted myotubes by in vitro transfection Primary myotubes by in vitro transfection Tibialis anterior muscle by i.m. Tibialis anterior muscle by i.m.

Target muscles & delivery of AO

Primary myotubes by in vitro transfection PMOs Ex 52 & 53 Primary myotubes by in vitro transfection PMOs Ex 52 & 53 Tibialis anterior muscle by i.m. or i.p. 2¢OMePSs Ex 19–25 Immortalized myotubes by or 9 PMOs in vitro transfection PMOs Ex 19–25 Tibialis anterior muscle by i.m. 2¢OMePSs Ex 50 & 51 or Converted myotubes by in vitro Ex 51 & 52 transfection 2¢OMePSs Ex 19 & 20 or Primary myotubes by in vitro Ex 20 & 21 transfection

4 2¢OMePSs

10 vPMOs

10 vPMOs

Ex 45–51 & ex 53–55 Ex 45–51 & ex 53–55 Ex 45–51 & ex 53–55

10 vPMOs

Ex 6 & 8 Ex 6 & 8

3 2¢OMePSs 3 PMOs

mdx52 mice Deletion of ex 52

Ex 6 & 8

4 2¢OMePSs

2 2¢OMePSs Ex 6 & 8 or 2 PMOs 2 PMO-Peps Ex 6 & 8

Ex 6 & 8 Ex 6 & 8

3 PMOs 4 PMOs

3 or 4 vPMOs Ex 6 & 8

No. of AOs & AO chemistry

GRMD dogs

CXMD dogs A point mutation at ASS in intron 6

Samples + N.A. + N.A. N.A. N.A. +

+ N.D. N.A. + +

N.D. + + N.A. + N.A. N.A.

+ + + + + + +

+ + + + +

+ + + + + + +

N.A.

+ N.A.

N.A.

+

N.A.

N.A.

+

+

N.A.

N.A.

N.A.

+

N.A. N.A.

N.A.

+ +

+

Western ICC or RT-PCR blotting IHC

Dystrophin expression

Yokota et al, 2009a

Yokota et al, 2012b Saito et al. 2010

Yokota et al, 2012b

Reference

Aoki et al., 2012

N.A.

N.A.

(continued)

Adkin et al., 2012

Fall et al., 2006 Adkin et al., 2012

Fall et al., 2006

Mitrpant et al., 2009

Mitrpant et al., 2009

Amelioration of Aoki et al., 2012 pathology, improved grip power, no toxicity by blood test Mitrpant et al., 2009

N.A.

N.A. Yokota et al, 2009a Amelioration of Yokota et al, 2009a pathology Amelioration of Yokota et al, 2009a pathology, improved run ability, no toxicity by blood test McClorey et al., 2006a McClorey et al., 2006a Aoki et al., 2012

N.A.

N.A.

Treatment effects in vivo

Table 1. Summary of Preclinical Trials of Antisense Oligonucleotide-Mediated Multiple Exon Skipping

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3 2¢OMePSs

No. of AOs & AO chemistry

Target exons for skipping

Target muscles & delivery of AO

Ex 19 & 20 or Primary myotubes by in vitro Ex 20 & 21 transfection A point mutation at ASS 3 2¢OMePSs Ex 20 & 21 Primary myotubes by in vitro in ex 21 transfection A point mutation at ASS 2 2¢OMePSs Ex 21 & 22 Converted myotubes by in vitro in ex 22 transfection A nonsense mutation 2 2¢OMePSs Ex 17 & 18 Primary myotubes by in vitro of ex 17 transfection 20 bp deletion of ex18 2 2¢OMePSs Ex 17 & 18 Primary myotubes by in vitro transfection Duplication of ex 18 2 2¢OMePSs Ex 17 & 18 Primary myotubes by in vitro transfection Deletion of ex 7 4 PMOs Ex 6 & 8 Converted myotubes by in vitro transfection Deletion of ex 48–50 12 2¢OMePSs Ex 45–55 Primary myotubes by in vitro transfection Deletion of ex 46–50 12 2¢OMePSs Ex 45–55 Primary myotubes by in vitro transfection Duplication of ex 44 2 2¢OMePSs Ex 43 & 44 Converted myotubes by in vitro transfection Duplication of ex 52–62 2 2¢OMePSs Ex 52 & 62 Converted myotubes by in vitro transfection Deletion of ex 3–17 4 2¢OMePSs Ex 18, 19 & 20 Skeletal muscle fragments by ex vivo transfection Deletion of ex 46–50 2 2¢OMePSs Ex 17 & 51 Primary myotubes by in vitro transfection Deletion of ex 45, ex 2 2¢OMePSs Ex 42 & 55 Primary myotubes by in vitro 46–50 or ex 48–50 transfection Deletion of ex 51–55 2 2¢OMePSs Ex 45 & 59 Primary myotubes by in vitro transfection Deletion of ex 51–55 2 2¢OMePSs Ex 48 & 59 Primary myotubes by in vitro transfection A nonsense mutation 2 2¢OMePSs Ex 43 & 44 Converted myotubes by of ex 43 in vitro transfection Deletion of ex 46–50 2 2¢OMePSs Ex 45 & 51 Primary myotubes by in vitro transfection Deletion of ex 48–50 2 2¢OMePSs Ex 45 & 51 Primary myotubes by in vitro transfection

8 bp deletion in ex 20

Mutation

N.A. N.A. N.A. + + N.A.

N.D. N.D. N.D. + + +

N.D.

+

N.A.

N.D.

+

N.D.

+

+

N.A.

N.A.

+

+

N.A.

+

N.D.

N.A.

+

+

N.A.

+

+

N.A.

+

+

N.A.

+

N.A.

+

+

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

+

N.A.

N.A.

+

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

Western ICC or RT-PCR blotting IHC

Dystrophin expression Treatment effects in vivo

Aartsma-Rus et al., 2007 Aartsma-Rus et al., 2007 McClorey et al., 2006b Aartsma-Rus et al., 2006a Aartsma-Rus et al., 2006a Aartsma-Rus et al., 2006a Aartsma-Rus et al., 2006a Aartsma-Rus et al., 2004a Aartsma-Rus et al., 2004a Aartsma-Rus et al., 2004a

van Vliet et al., 2008

van Vliet et al., 2008

Saito et al. 2010

Forrest et al., 2010

Forrest et al., 2010

Forrest et al., 2010

Adkin et al., 2012

Adkin et al., 2012

Adkin et al., 2012

Reference

2¢OMePS, 2¢O-methylated phosphorothioate; AO, antisense oligonucleotide; ASS, acceptor splice site; CXMD, canine X-linked muscular dystrophy; DMD, Duchenne muscular dystrophy; Ex, exon; GRMD, golden retriever muscular dystrophy; i.m., intramuscular injection; i.v., intravenous injection; ICC, immunocytochemistry; IHC, immunohistochemistry; N.A., not available; N.D., not detected; PMO, phosphorodiamidate morpholino oligomer; PMO-Pep, PMO-conjugated to peptides; RT-PCR, reverse transcription polymerase chain reaction; vPMO, vivo PMO;

DMD patients

Samples

Table 1. (Continued)

MULTIPLE EXON SKIPPING FOR DMD

However, it is unclear how each truncated dystrophin protein differs in terms of its functions in the muscle membrane. To answer this question, extensive structure-function analyses of truncated dystrophin proteins, including deletion of the exon 45–55 region, are very reasonable approaches to predict the truncated proteins that result from exon skipping, as previously investigated with shortened-dystrophin constructs, which can express the truncated proteins without some spectrin repeats and hinge regions (Lai et al., 2009; Banks et al., 2010). The Potential of AO-Mediated Multiple Exon Skipping for DMD

Several promising approaches have been tested to try to rescue muscular dystrophies, such as gene therapy that uses virus vectors (Karpati and Acsadi, 1993; Yuasa et al., 1998; Sakamoto et al., 2002; Yoshimura et al., 2004; Quenneville et al., 2007; Ohshima et al., 2009; Koo et al., 2011) and transplantation research that uses bone-marrow stem cells, side population stem cells, mesoangioblasts, embryonic stem cells, and induced pluripotent stem cells (Bretag, 2007; Mooney and Vandenburgh, 2008). However, these strategies have been problematic, due to strong immune responses, difficulties in selecting adequate routes of administration, the efficacy of systemic transplantation, the limited amount of cells that can be obtained from a single biopsy, difficulties in promoting the proliferation of these cells, and the potential development of cancer. AO-mediated exon skipping is currently a most promising therapeutic approach for restoring dystrophin protein production using the patient’s own mutated dystrophin transcripts. The aim of exon skipping therapy is to improve severe DMD symptoms into the milder symptoms seen in BMD using AOs. AOs bind to complementary sequences of premature mRNA, and AO lengths of 25–31 base pairs are generally more effective than shorter AOs in promoting exon skipping (Harding et al., 2007). Such oligos are designed to target exon/intron boundaries or exonic splicing enhancer sites, thus interfering with spliceosome assembly and producing in-frame dystrophin mRNAs by removing exon(s) (Aartsma-Rus et al., 2006a; Yokota et al., 2009b, 2012a). AOs can be used for exon skipping for almost all internal exons (i.e., to skip the 77 exons from exons 2 to 78) (Wilton et al., 2007). However, it is unlikely that this therapy can be used to skip the first exon (exon 1) or the last exon (exon 79) without disrupting 5¢ capping or polyadenylation, respectively (Wilton et al., 2007; Yokota et al., 2012a). As described above, phase 2/3 clinical trials of AOs that target exon 51 are ongoing, but only 13% of all DMD patients can be treated using exon 51 skipping (Aartsma-Rus et al., 2009; Cirak et al. 2011; Goemans et al. 2011). Altogether, single exon skipping can be used to treat 64% of all DMD patients (Aartsma-Rus et al., 2009). In contrast, by targeting two or more exons of the DMD gene, multiple exon skipping therapy can theoretically be used to treat approximately 90%, 80%, and 98% of DMD patients with deletion, duplication, and nonsense mutations, respectively (Yokota et al., 2007b, 2012a; Aartsma-Rus et al., 2009). In contrast to deletion mutations, nonsense mutations are distributed throughout the gene (Malik et al., 2010). Therefore, a variety of exonspecific AOs are needed to cover most patients with nonsense mutations.

61

Notably, skipping the entire exon 45–55 region, which eliminates the ‘‘hotspot’’ region for deletion mutations of the DMD gene, has been suggested as an approach that could be used in more patients (Be´roud et al., 2007; Yokota et al., 2007a; Nakamura et al., 2008; van Vliet et al., 2008; Aartsma-Rus et al., 2009; Aoki et al., 2013). This approach could treat up to 63% of DMD patients with deletion mutations, and up to 45% of all DMD patients. In addition, this specific deletion is associated with remarkably mild phenotypes in patients (Be´roud et al., 2007; Nakamura et al., 2008). When a cocktail of 10 vivo-morpholinos (vPMOs) was systemically injected into mdx52 mice with the aim of skipping exons 45– 55, dystrophin expression was restored in bodywide skeletal muscles and there was amelioration of the histopathological features of the disease (Aoki et al., 2012). Further, no toxicity was detected in blood tests. However, the functional recovery was only marginal, probably due to the limited efficacy of the delivery. Nevertheless, multiple exon skipping therapy is a very promising approach that would expand the applicability of AO-based treatment. In addition, multiple exon skipping might be used to select deletions that optimize the functionality of the truncated dystrophin protein Development of AOs for Multiple Exon Skipping

Many types of AOs have been developed with various nucleotide substitutions and backbone chemical modifications with the aim of more effectively modulating RNA processing (Aartsma-Rus et al., 2004b; Wilton and Fletcher, 2011; Juliano et al., 2012; Ja¨rver et al., 2013; Lee and Yokota, 2013). Oligonucleotide modification is essential for resistance to nuclease degradation, efficient delivery into cells, and high binding affinity to mRNA (Seio et al., 2009; Nishida et al., 2010). Two types of oligonucleotide chemistry are used widely in exon skipping strategies. Antisense oligonucleotides modified with 2’O-methylphosphorothioate (2’OMePS) contain a 2’-modification of the ribose ring (van Deutekom et al., 2001, 2007; Heemskerk et al., 2009a, 2010; Goemans et al., 2011). Phosphorodiamidate morpholino oligomers (PMOs, morpholinos) are another type of synthetic DNA analog in which the nucleic acid bases are bound to morpholine moieties instead of to deoxyribose/ribose rings, and the phosphodiester backbone is replaced by a phosphorodiamidate linkage (Summerton and Weller, 1997; Gebski et al., 2003; Alter et al., 2006; Fletcher et al., 2006, 2007; Karkare and Bhatnagar, 2006; Kinali et al., 2009; Malerba et al., 2009; Popplewell et al., 2009; Yokota et al., 2009a; Goyenvalle et al., 2010; Wu et al., 2010, 2011b; Cirak et al., 2011; Malerba et al., 2011). These chemical modifications increase the stability and cellular uptake of AOs (Adams et al., 2007). The advantages of PMOs are particularly notable, as the nonionic backbone of PMOs helps reduce off-target effects while increasing water solubility and nuclease resistance (Hudziak et al., 1996; Summerton and Weller, 1997; Sazani et al., 2011). PMOs have a stronger binding affinity for RNA, and do not elicit an immune response through Toll-like receptors and the interferon system (Summerton, 2007). Because of these advantages, PMOs are preferred for use in multiple exon skipping in preclinical trials both in vitro and in vivo (Table 1). Although 2’OMePSs and PMOs are useful for exon

62

skipping therapy, further advances in AO modification might be required for multiple exon skipping because current AOs induce limited dystrophin expression. Recently, several new generation morpholino derivatives were developed with the aim of improving exon skipping efficiency. These derivatives include cell-penetrating peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), arginine-rich cell-penetrating peptide-conjugated PMOs (Pip-PMOs), and vivo-morpholinos (vPMOs) that contain cell-penetrating moieties composed of octa-guanidinium groups (Morcos et al., 2008; Moulton and Moulton, 2010; Yin et al., 2011a; Betts et al., 2012). The new generation morpholino derivatives have been reported to induce prolonged and extensive rescue of dystrophin expression in dystrophic dogs and mice (Jearawiriyapaisarn et al., 2008, 2010; Yin et al., 2008, 2009, 2010, 2011b; Wu et al., 2009, 2011a; Betts et al., 2012; Moulton, 2012; Yokota et al., 2012b). Multiple exon skipping has been achieved for exons 45–55 in the bodywide skeletal muscles of dystrophic mdx52 mice using a cocktail of antisense vPMOs that target each exon from exon 45 to exon 55 (Aoki et al., 2012). In contrast, multiple exon skipping using a 2¢OMePS cocktail did not seem to induce exons 45–55 skipping in two DMD patient cell lines with deletion mutations in exons 48–50 and exons 46–50 (van Vliet et al., 2008). However, these results cannot be compared because the studies used different types of cells and different AO designs. A possible strategy for optimizing multiple exon skipping is to prevent formation of hairpin loops and cross- and self-dimerization (or potential multimerization) between the synthetic AOs (Aartsma-Rus, 2012). In fact, cocktail 10 AOs that are designed so that self annealing and cross annealing are minimized effectively have led to skipping the entire exons 45–55 region in the mdx52 mouse model (Aoki et al., 2012). The Dystrophic Dog Model and Preclinical Development of Multiple Exon Skipping Therapies

Golden retriever muscular dystrophy (GRMD) is a spontaneous fatal canine disease and a homologue of human DMD (Kornegay et al., 1988; Sharp et al., 1992; Miyazato et al., 2011). Dr. Takeda’s group developed the canine X-linked muscular dystrophy model in Japan (CXMDJ), a strain of medium-sized dystrophic beagles, by artificial insemination of spermatozoa from a dog with GRMD into carrier female dogs (Shimatsu et al., 2003, 2005; Nakamura and Takeda, 2011). The pathological and clinical phenotypes of the dystrophic dog model are characterized by progressive muscle degeneration, including degeneration of the skeletal and cardiac muscles; fibrosis; shortened life span; and respiratory and cardiac failure. These characteristics make the dystrophic dog model more similar to DMD than any of the dystrophic mice models (Nguyen et al., 2002; Nakamura and Takeda, 2011). Dystrophic dogs develop severe cardiomyopathy, including vacuole degeneration in the Purkinje fibers, and show deep distinct Q-waves on electrocardiograms of the left ventricular wall that are similar to those in human DMD/ BMD patients (Nomura and Hizawa, 1982; Finsterer and Stollberger, 2001; 2003; 2007; Stollberger and Finsterer, 2005; Yugeta et al., 2006; Urasawa et al., 2008). One of the leading causes of early death in DMD is cardiac failure as-

ECHIGOYA AND YOKOTA

sociated with cardiomyopathy. However, the pathogenic mechanism remains unclear, and there is no effective therapeutic approach for dystrophin-deficient cardiac muscles. The dystrophic dog model could help resolve these issues as it provides much more information than the mouse model, which does not show severe cardiomyopathy. The dystrophic dog model is proving useful in preclinical antisense cocktail drug development (McClorey et al., 2006a; Yokota et al., 2011). Notably, except for mdx4cv mice, mouse models of DMD do not require multiple exon skipping (Chamberlain et al., 1987; Chapman et al., 1989; Danko et al., 1992; Mitrpant et al., 2009). As a result, there is much less information regarding in vivo multiple exon skipping in preclinical trials than information about single exon skipping (Table 1). In vivo studies in dystrophic dogs continue to be conducted in order to develop an efficient and safe method for AO delivery. The similarity between human and canine dystrophin sequences is another advantage of the dystrophic dog model. For example, 97% of exon 6 and 95% of exon 8 in the dystrophin gene match at the nucleotide sequence level (Saito et al., 2010). The dystrophic dog model has a point mutation at the acceptor splice site of intron 6 in the dystrophin gene, leading to the exclusion of exon 7 followed by a premature stop codon in exon 8 (Fig. 1) (Sharp et al., 1992). Thus, this model requires double exon skipping of exons 6 and 8 to restore the reading frame (McClorey et al., 2006a). Previously we used systemic injections of a three-PMO cocktail to provide the first proof of concept of multiple exon skipping that targeted exons 6 and 8 in CXMDJ dogs (Yokota et al., 2009a). This systemic treatment resulted in dystrophin expression at therapeutic levels in bodywide skeletal muscles, accompanied by histopathological amelioration of the disease. Importantly, the dogs recovered their ability to run, and there was no obvious toxicity over the course of the 24-week study. In contrast to the excellent efficacy of the therapy in skeletal muscles, dystrophin expression was not induced at high levels in the dogs’ cardiac muscle. A cocktail of 4 PMOs, which was composed of the same three PMOs (ortholog) and a newly designed PMO for more efficient skipping of exon 8, was used in cells from a DMD patient that had a deletion mutation in exon 7, and treatment of the cells restored dystrophin protein expression (Saito et al., 2010). One major concern is that unmodified AOs does not always have sufficient efficacy in terms of multiple exon skipping. As mentioned above, the new generation of morpholinos, PPMO and vPMO, show effective rescue of dystrophin expression at lower dose in the dystrophic mdx mouse model in vivo (Wu et al., 2009, 2012). Based on these results, we designed a vPMO cocktail with three or four AO sequences and examined its efficacy after intramuscular injection into the limb muscles of CXMDJ dogs (Yokota et al., 2012b). This local treatment with the three-vPMO cocktail (400 mg of each AO) induced more extensive and prolonged dystrophin expression for up to 8 weeks than treatment with unmodified morpholinos with the same sequence (1.2 mg of each AO). In addition, higher efficiency of dystrophin protein expression was achieved with the optimized four-vPMO cocktails. A series of preclinical trials using CXMDJ dogs and DMD patient cell lines has demonstrated the therapeutic potential of AO-mediated multiple exon skipping.

MULTIPLE EXON SKIPPING FOR DMD Progress to Date and Challenges for Clinical use of Multiple Exon Skipping Therapy

The studies reporting on the preclinical development of multiple exon skipping therapy that targets the DMD gene are summarized in Table 1. The first demonstration of multiple exon skipping using an AO cocktail was reported by Aartsma-Rus et al., in human DMD cells (Aartsma-Rus et al., 2004a). To date, studies demonstrated that AO cocktails could be used to skip the following: exons 6 and 8, exons 17– 18, exons 18–20, exons 19–20, exons 19–25, exons 20–21, exons 21–22, exons 43–44, exons 45–51, exons 45 and 51, exons 45–55, exons 50–51, exons 51–52, exons 52–53, and exons 52 and 62 (Aartsma-Rus et al., 2004a, 2007; Fall et al., 2006; McClorey et al., 2006a, 2006b; van Vliet et al., 2008; Mitrpant et al., 2009; Yokota et al., 2009a, 2012b; Forrest et al., 2010; Saito et al., 2010; Adkin et al., 2012; Aoki et al., 2012). These preclinical tests demonstrated skipping of multiple exons at the mRNA level, but dystrophin protein expression either was not shown or was not detectable by western blotting and/or immunohistochemistry or immunocytochemistry in most of these studies, especially in human DMD cell lines. Most of these studies used 2’OMePS AOs. The use of modified AOs, such as PPMOs and vPMOs, might improve restoration of dystrophin protein expression both in vitro and in vivo. The systemic rescue of dystrophin protein in bodywide skeletal muscles has been demonstrated in dystrophic dogs (skipping of exons 6 and 8 using a cocktail of PMOs) and in mdx52 mice (skipping of exons 45–55 using vPMOs) (Yokota et al., 2009a; Aoki et al., 2012). Although AO-mediated multiple exon skipping is a very attractive approach for expanding the therapeutic coverage of DMD patients, more preclinical studies are needed for rational development of this approach prior to human clinical trials. Currently, multiple exon skipping using AO cocktails faces several hurdles. The first major hurdle is its insufficient efficacy in terms of restoring dystrophin expression in bodywide muscles, especially in cardiac muscles, due to the poor efficiency of systemic AO delivery. Advances in AO chemistry have facilitated the restoration of dystrophin expression both in vitro and in vivo, but the levels of dystrophin protein induced by skipping many exons, such as exon 45–55 skipping, might be still lower than the desired therapeutic levels. To overcome this issue, it may be necessary to have a better understanding of the RNA splicing machinery. For example, understanding the order of DMD intron removal between exons 45–55 during splicing might help optimize the design of a cocktail of AOs with fewer oligos that can be used to generate effective exon 45–55 skipping. Second, unknown long-term toxicity is one of the major concerns of multiple exon skipping. To date, systemic multiple exon skipping has been reported with 3-PMO cocktails and 10-vPMO cocktails in the dystrophic dog and mouse models, respectively (Yokota et al., 2009a; Aoki et al., 2012). Cell-penetrating derivatives will increase the potency, although this also comes with a higher risk of side effects. So far, no toxicity has been detected at therapeutic dosages of vPMOs (6 mg/kg) and PPMOs (up to 30 mg/kg) in mice receiving systemic exon skipping treatment (Wu et al., 2009; Wu et al., 2012). Our very recent study also found no toxicity in blood tests after five biweekly injections of a 10-vPMO cocktail in mice (12 mg/kg each injection in total of 10 oligos) (Aoki et al.,

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2012). However, a single systemic injection of a high dose of PPMOs (150 mg/kg) in rats caused adverse events such as lethargy, weight loss, and elevated levels of blood urea, nitrogen, and serum creatinine (Amantana et al., 2007). In monkeys, systemic weekly injection of PPMOs at 9 mg/kg for 4 weeks led to mild tubular degeneration in the kidneys (Moulton and Moulton, 2010). In multiple exon skipping therapy, potential toxicity or off-target effects might be confounded, because repeated administration of high-dose antisense cocktail with many different AOs will be required to maintain the therapeutic effect during the patient’s lifetime. Thus, it is critical to think about longer-term effects when assessing AO therapies in preclinical testing. Ideally, multiple exon skipping should be induced using as few AOs as possible. This strategy has been tested by using two AOs to exclude the entire exon 17–51 region plus exons 42–55, 45– 59, and 48–59 (Aartsma-Rus et al., 2006a). However, the strategy did not excise the intended exons, illustrating the complexity of this approach. Third, the regulatory stance of federal agencies, such as the United States Food and Drug Administration, on combination antisense drug cocktails is not clear. Currently, newly developed AOs that target different sequences are considered by regulatory authorities to be separate novel drugs. Cocktails of AOs could potentially be considered a single drug by major regulatory agencies in the European Union, United States, and Japan (Aoki et al., 2013). If such cocktails are not considered a single drug, the required preclinical toxicology studies will become extremely complicated and will hamper the translation of this approach. In contrast to the prospective preclinical trials, clinical studies of exon skipping therapy are currently at a critical moment. Very recently, GSK and Prosensa announced that the phase 3 clinical trial of drisapersen (a 2’OMePS AO that is 20 bp in length, GSK2402968/PRO051), which induces exon 51 skipping, did not meet the primary endpoint of statistically significant improvement in the Six-Minute Walking Distance test compared to the placebo group. In this clinical trial, weekly drisapersen treatment (6 mg/kg/week) was administered to DMD patients by subcutaneous injection for 48 weeks. There seems to be plenty of room for improvement in this trial. One possible reason that the clinical trial may have failed is that exon 51 might not be an ideal target for exon skipping. Exon 51 skipping is theoretically applicable to the highest percentage (13%) of DMD patients, a subgroup of patients with deletion mutations, such as those with deletion mutations in exons 50, 45–50, 47–50, 48–50, 49–50, and 52. This indicates that distinct truncated dystrophin proteins can be produced in individual patients who receive the same treatment. However, approximately half of patients with inframe deletion mutations that start or end at exon 51 are reported to develop severe DMD on the Leiden Open Variation Database (LOVD) (Aartsma-Rus et al., 2006b; Yokota et al., 2007a). In terms of the incidence of BMD, other exons such as exon 45, 53, or 55 could be more promising targets for improving function of the skeletal muscles. The single exon skipping that targets exon 51 might be changed to multiple exon skipping to induce more functional dystrophin protein. Clinical evidences from the LOVD show that a large deletion between exons 45–55 is associated with milder BMD forms in more than 95% of all cases (Be´roud et al., 2007), although multiple exon skipping is still technically challenging.

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A second reason that the clinical trial may have failed is the chemical structure of the drug. In particular, the efficiency of exon skipping with 2’OMePS backbone chemistry is reported to be lower than that with PMOs, especially in vivo (Heemskerk et al., 2009b; Yokota et al., 2009a). In addition, the shorter AO, which was 20 bp in length, might not have been effective as well as a longer construct in terms of exon skipping (Harding et al., 2007). Finally, skipping efficiency via subcutaneous administration is 40%–80% lower than that achieved via intravenous administration, at least in the mouse model (Heemskerk et al., 2010). Taken together, exon skipping therapy that targets different exons, or with different AO chemistry and intravenous administration might be more effective in terms of recovery of motor function in DMD patients. This may be achievable in the near future. Conclusions

AO-mediated exon skipping is a promising therapeutic approach for treating DMD, and multiple exon skipping could rescue most DMD patients, regardless of mutation type. The proof of concept of systemic multiple exon skipping has been provided in dystrophic dog and mouse models using 3-PMO and 10-vPMO cocktails, respectively. However, multiple exon skipping remains technically challenging. For systemic multiple exon skipping therapy to succeed, more effective systemic delivery methods are needed and the long-term toxicity of cocktail AOs needs to be determined. Achieving effective multiple exon skipping with fewer AOs would accelerate the translation of this approach for clinical use. Acknowledgments

This work was supported by The University of Alberta Faculty of Medicine and Dentistry, Parent Project Muscular Dystrophy (USA), The Friends of Garrett Cumming Research Funds, HM Toupin Neurological Science Research Funds, Muscular Dystrophy Canada, Canada Foundation for Innovation, Alberta Ministry of Enterprise and Advanced Education, the Women and Children’s Health Research Institute (WCHRI), and Department of Defense: W81XWH09-1-0599. Author Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Toshifumi Yokota, PhD Department of Medical Genetics University of Alberta Faculty of Medicine and Dentistry 8812 112th Street Edmonton, Alberta, T6G 2H7 Canada E-mail: [email protected] Received for publication September 11, 2013; accepted after revision November 26, 2013.