Skip to main content
NCBI home page
Artificial DNA, PNA & XNA logoLink to Artificial DNA, PNA & XNA
. 2011 Jan-Mar;2(1):6–15. doi: 10.4161/adna.2.1.15425

Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

Camilla Brolin 1, Takehiko Shiraishi 2,✉
PMCID: PMC3116580  PMID: 21686247

Abstract

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2′-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA).

Key words: antisense, DMD, exon skipping, in vivo, splicing modulation, therapy

Introduction

Duchenne Muscular Dystrophy (DMD), an X-linked recessive disorder, is the most common form of muscular dystrophy that affects one in every 3,500 newborn boys. DMD is characterized by progressive muscle wasting, cardiomyopathy, respiratory failure and premature death. Affected boys are diagnosed between the ages of 3 and 5, restricted to a wheel chair by the age of 13, and die of cardiac and/or respiratory failure before their third decade. DMD is caused by mutations in the dystrophin gene,1 derived from deletions (65%), duplications (15%) or nonsense and other small mutations (20%). All these mutations result in the disruption of the open reading frame and ultimately absence of functional dystrophin. Dystrophin proteins crosslink the extracellular connective tissue to the intracellular actin filament network via protein complexes at the sarcolemma. A lack of this connective protein results in severely weakened muscle cells and loss of muscle functions accompanied by muscle tissue replacement by fatty acid and connective tissue. A number of molecular therapies are under intensive investigation to restore the dystrophin expression. Among the novel molecular therapeutic approaches, “exon skipping” is among the most promising methods.2 This method uses synthetic antisense oligonucleotides (AOs), targeted to specific regions of pre-mRNA transcripts, to modulate splicing of pre-mRNA thereby causing skipping of specific exon(s) and correction of the reading frame to yield functional dystrophin. There has been a considerable progress using animal DMD models employing both local and systemic administrations of AOs. In general, all approaches showed some success with the restoration of dystrophin expression albeit a large variety of efficacy with certain organs/tissues depending on factors including type of AO chemistry used and modifications for delivery. Recently, studies using cell penetrating peptides as carrier for delivery showed successful exon skipping in all muscle tissues investigated including heart tissue.3–5 Moreover, two recent clinical trials provide realistic hope for the development of DMD treatment because two separate trials (mainly in UK and Netherland) using different AOs chemistries showed safe and promising results with both localized and systemic delivery.6,7 Here we review the recent progress and challenges in the antisense induced splice-modulation studies for DMD in vivo using animal models.

Exon Skipping Therapy in DMD

In DMD patients and DMD animal models, dystrophin is basically absent at the sarcolemma, although there are very few dystrophin-positive fibers, so called “revertant fibers”. Currently, it is thought that the revertant fibers are produced through the intrinsic alternative splicing processes that sometimes cause skipping of mutated exon(s), restoration of the open reading frame and expression of functional dystrophin.8 This phenomenon has been exploited for artificial splicing modulation by use of AOs. AO-mediated exon skipping modulates pre-mRNA splicing in the way that out of frame mutations are converted into the in-frame transcripts through the skipping of one or multiple exons to yield in-frame but internally truncated transcripts translatable into functional dystrophin proteins analogous to those found in a clinically milder form of Becker muscular dystrophy (BMD).9 AOs have been designed to target pre-mRNA exons at or in close proximity to the site of mutation such as exon/intron boundary, exon splice enhancer element or branch point. This is an appealing approach for conversion of the severe DMD disease into a condition analogue to the substantially milder BMD. This “Exon skipping principle” has been reported to be practical for up to 83% of all DMD patients including patients with deletion mutations (54%), small mutations (23%) and duplication (6%).10

Antisense Oligonucleotides (AOs) and Delivery

AOs are chemically synthesized short (typically 20–30 bp) single stranded nucleic acids designed to hybridize with target RNA sequences to modulate gene expressions via various processes including splicing modulation. Various AO chemistries have been developed to improve their properties such as specific sequence binding, nuclease stability, safety. These include bicyclic-locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), 2′-O-methyl phosphorothioate AO (2OME-PS), peptide nucleic acid (PNA), phosphorodiamidate morpholino oligomer (PMO).11 Among the tested chemistries so far, three AOs, namely 2OME-PS, PMO and PNA (Fig. 1), have been used for exon skipping in DMD animal models (Table 1). Moreover, two of these, 2OME-PS and PMO have been tested in clinical trials targeting DMD exon 51. Other AO chemistries with highly efficient exon skipping properties may offer advantages especially for in vivo applications. Though various issues must be addressed with AOs therapy including, delivery, efficacy and toxicity, however, ethical hurdles might be reduced as compared with gene therapy using viral vectors or stem cell transplantation because AOs are classified as classical drugs by FDA and other counterparts rather than a gene therapeutic agent.

Figure 1.

Figure 1

Open in a new tab

Chemistries of antisense oligomers. (A) 2′-O-methyl phosphorothionate (2OME-PS); (B) Phosphorodiamidate morpholino oligomer (PMO); (C) Peptide nucleic acid (PNA).

Table 1.

Exon skipping studies in animal models

Animal model1 (year) AOs2 Tested age Target exon(s) skipping AO modification/delivery method3 Administration4, dose Effects5 Ref. (year)
mdx mice
2001
2OME-PS 3 weeks old 23 Lipofectin 2–4 × 1 µg/weekly i.m. (QUAD) Exon skipping and dystrophin expression (IH, WB) 13
2003
2OME-PS 2, 4 weeks and 6 months 23 F127 3 µg (2, 4 weeks mice) or 9 µg (6 months mice) i.m. (TA) Exon skipping and dystrophin expressing (IH), 20% of wt level (WB). Improvement of max. isometric tetanic force 31
11–18 weeks old 23 Electroporation 8 µg i.m. (TA) Exon skipping and dystrophin expression (IH, WB) 32
PMO 3 weeks old 23 Lipofectin/leash 1 µg i.m. (TA) Exon skipping and dystrophin expression (IH, WB) 33
2004
2OME-PS 6 weeks old 23 PEI 2 × 25 µg/weekly i.m. (TA) Dystrophin expression (IH) 34
2005
2OME-PS 3, 6 weeks and 6 months old 23 F127 2 mg or 3 × 2 mg/weekly i.v. Exon skipping and dystrophin expression in all muscles except heart (IH, WB only for 3 injections) 22
PS 5 weeks old 19 0.2, 2, 20, 200 mg/kg i.p. Exon skipping in femoral muscle (20 mg/kg) 35
2006
2OME-PS 6–8 weeks old 23 PEG-PEI 5, 20 µg or 2 × 20 µg/weekly i.m. (TA) Dystrophin expression (IH), 2–5% of wt level (WB) 36
PMO 3, 6 weeks and 6 months old 23 Lipofectin/leash 2, 10 µg i.m. (TA). Exon skipping and dystrophin expression in multiple muscles (IH, WB) and improvement of max. isometric tetanic force (i.v., 7*2 mg) 23
2 mg, 3 × 2 mg/weekly or 7 × 2 mg/weekly i.v.
11 days and 16 weeks old 19–25 used as cocktail 2, 10 µg i.m. (TA) Multiple exon skipping and dystrophin expression (IH, WB) 37
2OME-PSs, PMOs, PNAs neonatal, 6 and 36 weeks old 23 F127/leash 2, 5, 10 µg i.m. (TA). i.m.: 2OME-PS, very low exon skipping (IH); PNA, no exon skipping (IH); PMO, exon skipping and dystrophin expression (IH, WB). Multiple i.p. injection, low levels of exon skipping in multiple muscles and dystrophin expression in TA, diaphragm and ilium (IH, WB) 38
25 mg/kg or 7 × 25 mg/kg + 2 × 3 × 25 mg/kg i.p.
2007
PMO neonatal, 4 weeks and 1 years old 23 (RXR)4XB 1–25 mg/kg or 4 × 1, 2 or 5 mg/kg/weekly i.p. Exon skipping and dystrophin expression in all muscles tested except heart by 4 weekly injections (5 mg/kg) (IH, WB). Reduced dystrophic pathology 39
2008
2OME-PS 2 months old 23 Lipofectin 15 × 6 µg/monthly i.m. (paraspinal muscles) Reduction in kyphosis, centronucleation and fibrosis. Increased dystrophin expression (IH) 40
6–9 weeks old 23 PEG-PEI 3 × 1, 5, 10 or 20 µg/weekly or 10*1 or 5 µg/4 days between injections i.m. (TA) Exon skipping and dystrophin expression (IH), up to 20% of wt levels (WB) 41
PMO 7–8 weeks old 23 (RXRRBR)2XB 4 × 12 mg/kg/daily i.v. Exon skipping and dystrophin expression bodywide and in cardiac muscle (IH, WB) 42
12 months old 23 F127 100 µg or 2 × 100 µg/weekly i.a. (femoralis). Exon skipping and dystrophin expression (IH, WB), force recovery in EDL (i.a.). Exon skipping with i.c. is less efficient than i.m. (IH) 43
10 or 30 µg i.c.
15 µg i.m. (TA)
4–5 weeks and 4 months old 23 (RXRRBR)2XB 2 µg i.m. (TA). 30 mg/kg or 6 × 30 mg/kg/biweekly i.v. Exon skipping and dystrophin expression bodywide and in cardiac muscle (IH, WB). Functional improvement in skeletal- (grip strength, rotarod test) and cardiac muscle 3
6–8 weeks old 23 (RXR)4XB, (RXRRBR)2XB 25 mg/kg or 3 × 6 mg/kg/weekly i.v. Exon skipping and dystrophin expression bodywide and in cardiac muscle (IH, WB). Functional improvement in muscle (grip strength) 44
PNA 8 weeks old 23 Pip1, Pip2a, Pip2b, (RXR)4 5 µg i.m. (TA) Exon skipping and dystrophin expression (IH) 45
2OME-PS, PMO, PNA 3 weeks, 2 and 5 months old 23 TAT, MSP, AAV6, AAV8 2, 5 or 10 µg i.m. (TA) Exon skipping and dystrophin expression (IH, WB) 46
2009
2OME-PS 6–8 weeks old 23 Polymersomes 5 µg i.m. (TA) Exon skipping and dystrophin expression (IH) 47
8–10 weeks old 23 PMMA nanoparticles 3 × 0.9 µg/kg/weekly i.p. Exon skipping and dystrophin expression bodywide (IH, WB) and in cardiac muscle (IH) 48
6–8 weeks old 23 PEG-PEI, PLGA-PEG-PEI 3 × 5 µg/3 days between injections i.m. (TA) Exon skipping and dystrophin expression (IH), 5–10% of wt (WB) 49
2009
PMO 6 months old 23 2 µg i.m. (TA). Multiple i.v. injections of low doses (4 × 5 mg/kg) show more exon skipping and dystrophin expression than a single dose of the same total amount (IH, WB) 50
1–200 mg/kg or 4 × 5 or 50 mg/kg/weekly i.v.
4–5 week old mdx mice 23 Non-peptide dendrimeric octaguanidine 2 or 10 µg i.m.. Exon skipping and dystrophin expression bodywide (>50% of wt level (WB)) and in cardiac muscle (10%, WB) with 5 × 6 mg/kg biweekly i.v.. Improvement of muscle pathology 51
6, 30 or 10 × 15 mg/kg every other day i.p..
6 mg/kg or 5 × 6 mg/kg/biweekly i.v.
6–8 weeks old 23 AR-XB, MSP, AR-MSP, MSP-AR 25, 30, 40 mg/kg or 6 × 3 or 3 × 6 mg/kg/weekly i.v. High level exon skipping and dystrophin expression bodywide, only low levels in heart with B-MSP-PMO (IH, WB). Improvement of muscle pathology and function (grip strength) 52
2010
2OME-PS 6 weeks old 23 PMMA nanoparticles 7 × 225 µg/weekly i.p. Exon skipping and dystrophin expression in all muscles including smooth muscle (IH, WB) 53
4–5 weeks old 23 12.5 mg/kg or 2*20 µg i.m. (GC). Exon skipping and dystrophin skipping in all muscles including smooth muscle in skin with i.v., i.p. and s.c. administration. Similar bioavailability for all administration routes 54
50 mg/kg, 3*100 mg/kg/every other day or 5 × 250 mg/kg/daily i.v..
4–8 × 2 × 25, 50 or 100 mg/kg/weekly, 5 × 250 mg/kg/daily s.c..
5 × 250 mg/kg/daily i.p.
PMO 6–8 weeks old 23 Guanine analogues 2 µg PMO + 1 or 10 µg guanine analogues i.m. (TA) Enhanced exon skipping and dystrophin expression with guanine analogues (IH, WB) 55
8–16 weeks old 23 (RXRRBR)2XB 2 × 4 × 12 mg/kg/daily with a two week interval i.v. Exon skipping and dystrophin expression in skeletal, smooth and cardiac muscles (IH, WB). Improvement of cardiomyopathy 5
2010
4–5 weeks old 23 0.03–3 g/kg i.v. Dose dependent exon skipping and dystrophin expression. 50% of wt level in skeletal muscles and 30% in heart with 3 g/kg (WB). Improved muscle pathology 56
6–8 weeks old 23 AR-MSP-X 3, 6 mg/kg or 6 × 3 or 6 mg/kg/biweekly i.v. Almost complete exon skipping and dystrophin expression (IH), 50% of wt (WB) in all muscles except heart. Improvement of muscle pathology and function (grip strength) with 6 mg/kg multiple injections 4
PNA 2 and 12 months old 23 MSP, AVV6, R9F2, (RXR)4, Pip2b 5 µg i.m. (TA). No effect with CPP-conjugates and long lasting (−16 week) exon skipping and dystrophin expression with unmodified PNA (i.m.). Highest level of exon skipping and dystrophin expression with longest PNA (25 mer, i.v.) 57
25, 50, 100 mg/kg or 3 × 15 mg/kg i.v.
2OME-PSs, PMOs, PNAs 6–8 weeks old 23 TAT(RNA), Pip2B, (RXR)4 (PNA and PMO), AR-X, MSP-X-AR, AR-MSP-X (PMO) 5 µg i.m. (TA) Exon skipping and dystrophin expression (IH). High correlation between in vitro and in vivo data 25
2011
PMO 9 months old 23 10, 20, 40 and 60 µl (1 µg/µl) i.m. (TA) Dystrophin expression (IH, WB). High correlation between the number of dystrophin positive fibers and improved resistance to lengthening contraction. 58
Other animal models
2004
hDMD mice, mdx mice, normal 2OME-PSs 46 (normal, mdx mice) 44–49 (hDMD mice) PEI, SAINT 0.9–5.4 nmol (normal mice), 0.36–3.6 nmol (mdx mice)or 3.6 nmol (hDMD mice) i.m. (GC) Normal mice, exon skipping. Mdx mice, increased exon skipping in regenerating fibers. hDMD mice, human DMD exon 44, 46 and 49 skipping (IH). 16
2007
hDMD mice PMOs 5 weeks old 51 2 × 2.9 nmol/daily i.m. (GC) Exon skipping 59
2009
hDMD mice and mdx mice 2OME-PSs, PMOs 4–5 weeks old 23 (mdx), 44,45,46 and 51 (hDMD) 2 × 20 µg/daily i.m. (GC). Mdx mice, i.m. and i.v. injections with PMO showed higher levels of exon skipping than 2OME-PSs. hDMD mice, PMO and 2OME-PS showed comparable levels of exon skipping (IH, WB) 24
3× 3 × 100 mg/kg/weekly i.v. (mdx mice only)
4CV mice 2OME-PSs, PMOs 52/53 Used as singular or cocktail, F127 (2OME-PS) 100 µg i.m. or i.v. (2OME-PS). 40 µg i.m. (PMO) Exon skipping and dystrophin expression with PMO-cocktail only (IH), 5–7% of wt level (WB) 18
DMD dog model 2OME-PSs, PMOs 0.5–5 years old 6–9 used as cocktail 0.12–1.2 mg i.m. (TA, ECU). Exon skipping and dystrophin expression (i.m., IH, WB). Bodywide dystrophin expression (average 26%, less in heart), reduced inflammatory signals, improved timed running tests and clinical symptoms (i.v., PMO cocktail) 19
5–11 × 120–200 mg/kg/weekly or biweekly i.v.
2010
mdx52 mice PMOs 8 weeks old 51 1–10 µg i.m. (TA). Exon skipping and dystrophin expression in all muscles except heart, 20–30% of wt level (WB). Improvement of muscle pathology and function with multiple injections 17
80, 160, 320 mg/kg or 7 × 320 mg/kg/weekly i.v.
dKO mice PMO 10 days old 23 (RXR)4XB 6 × 5 or 25 mg/kg/weekly i.p. High levels of exon skipping and dystrophin expression bodywide except heart with 6 × 25 mg/kg (WB, IH). Improvement of muscle pathology and function (grip strength, force measurements) 15
hDMD mice PMOs 53 20 µg i.m. (GC) Screening of 24 PMOs in vitro. Exon skipping with 6 PMOs (in vivo) 60
Open in a new tab

Abbreviations:

1

dKO mice, Dystrophin/Utrophin double knockout mouse; DMD dog model [canine X-linked muscular dystrophy (CXMD) beagle dog], hDMD mice (transgenic human DMD mice), mdx52 mice (exon 52-deleted mice), 4CV mice [Cg-Dmd (mdx-4Cv)/J (4(CV)] mice carrying nonsense mutation in exon53).

2

AO, antisense oligonucleotide; 2OME, 2′-O-methyl; PMO, phosphorodiamidate morpholino; PNA, peptide nucleic acid; PS, phosphorothioate.

3

AAV6, TVAVNLQSSSTDPATGDVHVM; AAV8, IVADNLQQQNTAPQIGTVNSQ; AR, (RXRRBR)2 (where R is arginine, × is 6-aminohexanoic acid, B, β-alanine); F127, block co-polymer transfection; Lipofectin, cationic lipids; MSP (Muscle specific peptide), ASSLNIA; PEG, Polyethylene glycol; Pip1, (RXR)3-IKILFQNRRMKWKKC; Pip2a, (R-X-R)3-IdKILFQNdRRMKWHKBC; Pip2b, (RXR)3-IHILFQNdRRMKWHKBC; PLGA, poly(lactic-co-glycolic acid); PMMA, poly(methyl methacrylate); PEI, polyethylenimine; SAINT, cationic pyridinium transfection reagent; Tat, YGRKKRRQRRRP.

4

ECU, extensor carpi ulnaris; EDL, extensor digitorum longus; GC, gastrocnemius; i.a., intra arterial injection; i.c., intra cardiac injection; i.m., intra muscular injection; i.v., intravenous injection; i.p., intra penetrial injection; QUAD, quadriceps; s.c., Subcutaneous injection; TA, tibilias anterior.

5

IH, immunohistochemistry; WB, Western blot; wt, wild type.

Animal Models

Prior to clinical trials involving AOs, in vivo studies with animal models are indispensable. Such studies provide valuable information on both efficacy of exon skipping and improvement of muscle functions whereas in vitro studies are only able to asses the efficacy of exon-skipping. Convincing pre-clinical evidence for the therapeutic potential of DMD exon skipping has come from numerous in vivo studies in animal models of the disease (Table 1), most notably employing mdx mice.12 The mdx mice have a non-sense point mutation in exon 23, which creates a premature termination codon and results in a lack of dystrophin protein. The lack of the dystrophin causing muscle degeneration begins around three weeks of age, followed by continuing cycles of degeneration and regeneration though the mdx mice do not experience the same severe dystrophy as observed in human DMD patients. In vivo restoration of dystrophin expression by AOs was first demonstrated in mdx mice in 2001,13 and such mice showed recovery of dystrophin deleted for exon 23. Since then, there have been a considerable number of studies using mdx mice, investigating AOs with different chemistries both with local injection and systemic administration. In general, they all showed successful induction of target exon(s) skipping and improvement of dystrophin expression, though not all of the studies showed amelioration of muscle function (Table 1).

Human DMD is caused by a very heterogeneous set of mutations throughout the 79 exons spanning about 14 kb1,10 although there are two distinctive mutation hot-spots in the areas encompassing exon 3–7 and exon 45–55.14 For the development of AO therapies, each AOs chemistry and the target sequence must be validated in animal models as similar as possible to the human DMD targets in question. Recently, other animal models have been introduced such as dystrophin/utrophin double knockout mice,15 humanized DMD mice,16 mdx52 mice (carrying a deletion of exon 52 in murine DMD),17 4CV mice (carrying a nonsense mutation in exon53),18 and canine DMD model (mutation in exon 7).19 These transgenic mice provide indispensable tools for investigating the exon skipping strategies targeting above mentioned hot spot regions. Double knockout mice are relevant models in terms of pathology because of their severe disease progression whereas mdx mice show much milder and slower progression possibly owing to the overexpression of dystrophin homologue utrophin proteins. The dog DMD model in particular provides further advantages in relation to human DMD including a high similarity of target sites for exon skipping, a closer clinical phenotype with severe disease progression, and the requirement for skipping of multiple exons in order to restore the dystrophin reading frame.

Clinical Trial

Two European consortia are involved in clinical trials using different oligonucleotide chemistries [PRO051 (2OME-PS, Netherlands) and AVI-4658 (PMO, UK)]. Both target the skipping of exon 51 (Table 2). Safety study (phase 0) was completed in DMD patients and it showed exon skipping and partial restoration of de novo dystrophin following intramuscular injection of AOs [(tibialis anterior (TA) and extensor digitorum brevis (EDB) muscles respectively for PRO051 and AVI-4658] without adverse effects. In the case of PRO051,20 the 4 patients tested and showed up to 35% dystrophin positive fibers in biopsy samples after 28 days intramuscular TA administration (−8 mg) although there was no control of untreated starting muscle showing background dystrophin. In the case of AVI-4658,21 seven boys were injected with 0.09 or 0.9 mg in one of EDB muscles while contralateral muscle was used for injection control. Biopsy samples were taken from both EDB muscles on a blind basis 4 weeks after the treatment and used for analysis. This study showed that five out of seven patients with higher dosage exhibited de novo dystrophin expression and the mean intensity of dystrophin staining increased to an average of 26.4% of the controls. These two independent clinical trials were followed by repeated systemic administration studies (phase I/II and Ib/II) and both chemistries further confirmed the potential of exon skipping for DMD. A phase I/II dose-ranging safety study using PRO051 was performed on 12 patients. This demonstrated that PRO051 (5 weekly subcutaneous injection) was well tolerated up to 6 mg/kg and de novo dystrophin expression was detected in the patients with injections exceeding 0.5 mg/kg.6 AVI-4658 was further tested for open label phase Ib/II dose-ranging (0.5, 1, 2, 4, 10 and 20 mg/Kg) clinical trial with 12 weekly slow intravenous infusion administrations. The study, with 19 ambulatory patients, showed that AVI-4658 was well tolerated without serious adverse events. There was a substantial and novel dystrophin expression and dystrophin-positive fiber generation reaching up to 55%, although variable among patients, tended to be greatest in the highest two cohorts (www.avibio.com/). Taken together, both studies with repeated systemic administration demonstrate encouraging exon skipping and de novo dystrophin expression in a drug dose related manner, though full results of these trials have yet to be published. In addition, these AOs administrations were well tolerated so far by all patients. However, the effects of drug exposure for increased duration with repeated administrations should be examined because AOs have only a transient effect and they have to be repeatedly administered for the lifetime of the patient or until another treatment option becomes available. Currently, further multicentric phase III studies for both AOs chemistries and also additional multicentric phase I/II trials targeting exon 44 or 50 with 2OME-PSs are being planned.

Table 2.

Clinical trials'

Patients Mutations Target exon AO1 Administration2 Effects3 Ref
Four DMD patients Deletion of exon 50, 48–50, 49/50, 52 51 2MOE-PS 0.8 mg i.m. (TA) Exon skipping and dystrophin expression (3–12 % of normal level (WB), 64–97 of myofibers (IH)). No clinically apparent adverse events 20
7 DMD patients deletion of exon50, 48–50, 45–50, 49/50 51 PMO 0.09 or 0.9 mg i.m. (EDB) Increase of dystrophin staining intensity to 26. 4% (average, IH) of the healthy control muscle 21
One patient (10 years old) out-of-frame mutation and exon 20 deletion. 19 PS 4 × 0.5 mg/kg/weekly i.v. Exon 19 skipping and dystrophin expression (IH). 61
Open in a new tab

Abbreviations:

1

2OME-PS, 2′-O-methyl phosphonothioate; PMO, Phosphorodiamidate morpholino.

2

EDB, extensor digitorum brevis; i.m., intra muscular injection; i.v., intravenous injection; i.p., intra penetrial injection; TA, tibilias anterior.

3

IH, immunohistochemistry; WB, Western blot.

Issues of Exon Skipping Therapy and Current Challenges in DMD Splice Modulation

One limitation of AOs-mediated exon skipping strategy is a relatively poor and uneven efficacy in vivo. The degree of dystrophin restoration by systemically administered AOs is highly variable within and between muscles even after repeated i.v. injection.

A particularly low efficiency is observed for heart muscle as compared with skeletal muscle22,23 in mdx mice even at a high dose (<1–2% at 100 mg/kg24). This is probably because of poor cellular uptake of AOs within cardiac muscle tissue. Considering that many DMD patients die of cardiac complications, improvement of the AOs efficacy in cardiac muscles is very important. Also, to obtain more beneficial treatment (low dosage and fewer administrations) for the patients, it is indispensable to increase the efficacy of AOs to maintain adequate therapeutic levels of dystrophin protein in all muscles though what levels of dystrophin expression might be sufficient to induce clinical and functional improvement remains unknown. Recently, to improve the uptake and efficacy, peptide conjugated PMOs using arginine rich peptides such as (RXR)4, arginine rich peptide (AR as RXRRBR),25 were synthesized and reported to be more effective than native PMOs. Even combination with muscle specific peptide (MSP) as AR-MSP-PMO4 showed improved dystrophin splice correction in mdx mice. These peptide AOs conjugates are considerably potent as therapeutics, however, extensive safety test will be required before starting clinical trials as there is a concern about potential immune responses or possible toxicity due to the peptide moieties.

Another significant limitation of AO mediated DMD therapy is the fact that each AOs, developed for skipping of a specific exon, could be used only for a specific sub-set of mutation and hence only a subpopulation of DMD patients. For example, exon 51 skipping therapy, described above, may be feasible only up to 13% of all DMD patients.26 Including the next common mutations with exon 45, 53, 44 and 2,10,27,28 would still only enable targeting of 35% of all DMD patients. Analogously, Wood et al. estimated that approximately 40% of all patients could be targeted by single exon skipping using the most promising 10 AOs.29 To treat more patients by exon skipping strategy, elimination of two or more exons will be required. Theoretically single and double exon skipping would be applicable to 79% of deletions, 91% of small mutations and 73% of duplications amounting to 83% of all DMD mutations.10 However, to achieve this, at least 30 novel AOs will be required.29 Development of so many AOs as individual drugs will raise obvious issues related to costs and applicability. Currently, each AO is recognized as an individual drug and each undergoes rigorous safety and efficacy test prior to clinical test. This would be a significant hindrance for the development of high numbers of AOs because of the limited number of patients with a given mutation profile. Therefore, new legislative guidance and rational guidance from the regulatory representatives would be necessary to run the clinical trials in a realistic scale (fewer patients) with decent setup. Nonetheless, multi-exon skipping strategies have been tested using animal models such as dog DMD model and 4CV mice.18,19 These demonstrated restoration of dystrophin expression (exon 52/53 in mdx 4CV mice, exon 6/8 in DMD dog). Multi-exon skipping strategies, though conceptually attractive, tend to suffer from low efficiency.30

Conclusions and Future Prospects

At present, manipulation of pre-mRNA procession using the exon-skipping strategy is considered the most promising option for DMD treatment. Within the last few years, exon skipping therapy for DMD has rapidly advanced in vivo with new AOs and a number of animal models being developed. The data from two clinical trials in DMD patients, both targeting exon 51, demonstrated a realistic prospect of providing therapeutic benefits for 13% of patients.10 Though these preclinical/clinical data with AOs are encouraging, several issues still have to be addressed such as, delivery, efficacy and toxicity before these AOs and others can be further explored in clinical trials. Moreover, AOs are highly specific for the individual mutations and each AOs, target to specific exon skipping, would be relevant to only a few subsets of the patients. Therefore, there remains a need to develop larger number of AOs to enable targeting more DMD patients including subsets with rare DMD mutations. Less stringent clinical trial regulations would be needed to develop commercial “AO drugs for DMD” with such limited number of patients to provide beneficial therapy across the range of DMD patients.

References

  • 1.Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. 2006;34:135–144. doi: 10.1002/mus.20586. [DOI] [PubMed] [Google Scholar]
  • 2.van Ommen GJ, van Deutekom J, Aartsma-Rus A. The therapeutic potential of antisense-mediated exon skipping. Curr Opin Mol Ther. 2008;10:140–149. [PubMed] [Google Scholar]
  • 3.Wu B, Moulton HM, Iversen PL, Jiang J, Li J, Li J, et al. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci USA. 2008;105:14814–14819. doi: 10.1073/pnas.0805676105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Yin H, Moulton HM, Betts C, Merritt T, Seow Y, Ashraf S, et al. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO. Mol Ther. 2010;18:1822–1829. doi: 10.1038/mt.2010.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Jearawiriyapaisarn N, Moulton HM, Sazani P, Kole R, Willis MS. Long-term improvement in mdx cardiomyopathy after therapy with peptide-conjugated morpholino oligomers. Cardiovasc Res. 2010;85:444–453. doi: 10.1093/cvr/cvp335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Extance A. Targeting RNA: an emerging hope for treating muscular dystrophy. Nature reviews. 2009;8:917–918. doi: 10.1038/nrd3069. </unknown>. [DOI] [PubMed] [Google Scholar]
  • 7.Hammond SM, Wood MJ. PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy. Curr Opin Mol Ther. 2010;12:478–486. [PubMed] [Google Scholar]
  • 8.Lu QL, Morris GE, Wilton SD, Ly T, Artem'yeva OV, Strong P, et al. Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion. J Cell Biol. 2000;148:985–996. doi: 10.1083/jcb.148.5.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kunkel LM, Beggs AH, Hoffman EP. Molecular genetics of Duchenne and Becker muscular dystrophy: emphasis on improved diagnosis. Clin Chem. 1989;35:B21–B24. [PubMed] [Google Scholar]
  • 10.Aartsma-Rus A, Fokkema I, Verschuuren J, Ginjaar I, van Deutekom J, van Ommen GJ, et al. Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat. 2009;30:293–299. doi: 10.1002/humu.20918. [DOI] [PubMed] [Google Scholar]
  • 11.Nakamura A, Takeda S. Exon-skipping therapy for Duchenne muscular dystrophy. Neuropathology. 2009;29:494–501. doi: 10.1111/j.1440-1789.2009.01028.x. [DOI] [PubMed] [Google Scholar]
  • 12.Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis. 2008;31:1–19. doi: 10.1016/j.nbd.2008.03.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, et al. Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci USA. 2001;98:42–47. doi: 10.1073/pnas.011408598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Muntoni F. The development of antisense oligonucleotide therapies for Duchenne muscular dystrophy: report on a TREAT-NMD workshop hosted by the European Medicines Agency (EMA), on September 25th 2009. Neuromuscul Disord. 20:355–362. doi: 10.1016/j.nmd.2010.03.005. </unknown>. [DOI] [PubMed] [Google Scholar]
  • 15.Goyenvalle A, Babbs A, Powell D, Kole R, Fletcher S, Wilton SD, et al. Prevention of dystrophic pathology in severely affected dystrophin/utrophin-deficient mice by morpholino-oligomer-mediated exon-skipping. Mol Ther. 2010;18:198–205. doi: 10.1038/mt.2009.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ, Kaman WE, Janson AA, Vossen RH, et al. Targeted exon skipping in transgenic hDMD mice: A model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther. 2004;10:232–240. doi: 10.1016/j.ymthe.2004.05.031. [DOI] [PubMed] [Google Scholar]
  • 17.Aoki Y, Nakamura A, Yokota T, Saito T, Okazawa H, Nagata T, et al. In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther. 2010;18:1995–2005. doi: 10.1038/mt.2010.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Mitrpant C, Fletcher S, Iversen PL, Wilton SD. By-passing the nonsense mutation in the 4 CV mouse model of muscular dystrophy by induced exon skipping. J Gene Med. 2009;11:46–56. doi: 10.1002/jgm.1265. [DOI] [PubMed] [Google Scholar]
  • 19.Yokota T, Lu QL, Partridge T, Kobayashi M, Nakamura A, Takeda S, et al. Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Ann Neurol. 2009;65:667–676. doi: 10.1002/ana.21627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.van Deutekom JC, Janson AA, Ginjaar IB, Frankhuizen WS, Aartsma-Rus A, Bremmer-Bout M, et al. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med. 2007;357:2677–2686. doi: 10.1056/NEJMoa073108. [DOI] [PubMed] [Google Scholar]
  • 21.Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 2009;8:918–928. doi: 10.1016/S1474-4422(09)70211-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lu QL, Rabinowitz A, Chen YC, Yokota T, Yin H, Alter J, et al. Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in bodywide skeletal muscles. Proc Natl Acad Sci USA. 2005;102:198–203. doi: 10.1073/pnas.0406700102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD, et al. Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med. 2006;12:175–177. doi: 10.1038/nm1345. [DOI] [PubMed] [Google Scholar]
  • 24.Heemskerk HA, de Winter CL, de Kimpe SJ, van Kuik-Romeijn P, Heuvelmans N, Platenburg GJ, et al. In vivo comparison of 2′-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping. J Gene Med. 2009;11:257–266. doi: 10.1002/jgm.1288. [DOI] [PubMed] [Google Scholar]
  • 25.Wang Q, Yin H, Camelliti P, Betts C, Moulton H, Lee H, et al. In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy. J Gene Med. 2010;12:354–364. doi: 10.1002/jgm.1446. [DOI] [PubMed] [Google Scholar]
  • 26.Aartsma-Rus A, Janson AA, Kaman WE, Bremmer-Bout M, den Dunnen JT, Baas F, et al. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet. 2003;12:907–914. doi: 10.1093/hmg/ddg100. [DOI] [PubMed] [Google Scholar]
  • 27.Guglieri M, Bushby K. Molecular treatment in Duchenne muscular dystrophy. Curr Opin Pharmacol. 2010;10:331–337. doi: 10.1016/j.coph.2010.03.005. [DOI] [PubMed] [Google Scholar]
  • 28.Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard MT, et al. Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009;30:1657–1666. doi: 10.1002/humu.21114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wood MJ, Gait MJ, Yin H. RNA-targeted splice-correction therapy for neuromuscular disease. Brain. 2010;133:957–972. doi: 10.1093/brain/awq002. [DOI] [PubMed] [Google Scholar]
  • 30.van Vliet L, de Winter CL, van Deutekom JC, van Ommen GJ, Aartsma-Rus A. Assessment of the feasibility of exon 45–55 multiexon skipping for Duchenne muscular dystrophy. BMC Med Genet. 2008;9:105. doi: 10.1186/1471-2350-9-105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lu QL, Partridge TA. Antisense therapy corrects nonsense mutation by exon skipping. Discov Med. 2003;3:39–44. [PubMed] [Google Scholar]
  • 32.Wells KE, Fletcher S, Mann CJ, Wilton SD, Wells DJ. Enhanced in vivo delivery of antisense oligonucleotides to restore dystrophin expression in adult mdx mouse muscle. FEBS Lett. 2003;552:145–149. doi: 10.1016/S0014-5793(03)00904-9. [DOI] [PubMed] [Google Scholar]
  • 33.Gebski BL, Mann CJ, Fletcher S, Wilton SD. Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet. 2003;12:1801–1811. doi: 10.1093/hmg/ddg196. [DOI] [PubMed] [Google Scholar]
  • 34.Graham IR, Hill VJ, Manoharan M, Inamati GB, Dickson G. Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays. J Gene Med. 2004;6:1149–1158. doi: 10.1002/jgm.603. [DOI] [PubMed] [Google Scholar]
  • 35.Takeshima Y, Yagi M, Wada H, Matsuo M. Intraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle. Brain Dev. 2005;27:488–493. doi: 10.1016/j.braindev.2004.12.006. [DOI] [PubMed] [Google Scholar]
  • 36.Williams JH, Sirsi SR, Latta DR, Lutz GJ. Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers. Mol Ther. 2006;14:88–96. doi: 10.1016/j.ymthe.2005.11.025. [DOI] [PubMed] [Google Scholar]
  • 37.Fall AM, Johnsen R, Honeyman K, Iversen P, Fletcher S, Wilton SD. Induction of revertant fibres in the mdx mouse using antisense oligonucleotides. Genet Vaccines Ther. 2006;4:3. doi: 10.1186/1479-0556-4-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Wilton SD. Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide. J Gene Med. 2006;8:207–216. doi: 10.1002/jgm.838. [DOI] [PubMed] [Google Scholar]
  • 39.Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Steinhaus JP, et al. Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse. Mol Ther. 2007;15:1587–1592. doi: 10.1038/sj.mt.6300245. [DOI] [PubMed] [Google Scholar]
  • 40.Laws N, Cornford-Nairn RA, Irwin N, Johnsen R, Fletcher S, Wilton SD, et al. Long-term administration of antisense oligonucleotides into the paraspinal muscles of mdx mice reduces kyphosis. J Appl Physiol. 2008;105:662–668. doi: 10.1152/japplphysiol.00068.2008. [DOI] [PubMed] [Google Scholar]
  • 41.Williams JH, Schray RC, Sirsi SR, Lutz GJ. Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice. BMC Biotechnol. 2008;8:35. doi: 10.1186/1472-6750-8-35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S, et al. Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther. 2008;16:1624–1629. doi: 10.1038/mt.2008.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Vitiello L, Bassi N, Campagnolo P, Zaccariotto E, Occhi G, Malerba A, et al. In vivo delivery of naked antisense oligos in aged mdx mice: analysis of dystrophin restoration in skeletal and cardiac muscle. Neuromuscul Disord. 2008;18:597–605. doi: 10.1016/j.nmd.2008.05.011. [DOI] [PubMed] [Google Scholar]
  • 44.Yin H, Moulton HM, Seow Y, Boyd C, Boutilier J, Iverson P, et al. Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet. 2008;17:3909–3918. doi: 10.1093/hmg/ddn293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ivanova GD, Arzumanov A, Abes R, Yin H, Wood MJ, Lebleu B, et al. Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res. 2008;36:6418–6428. doi: 10.1093/nar/gkn671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Yin H, Lu Q, Wood M. Effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice. Mol Ther. 2008;16:38–45. doi: 10.1038/sj.mt.6300329. [DOI] [PubMed] [Google Scholar]
  • 47.Kim Y, Tewari M, Pajerowski JD, Cai S, Sen S, Williams JH, et al. Polymersome delivery of siRNA and antisense oligonucleotides. J Control Release. 2009;134:132–140. doi: 10.1016/j.jconrel.2008.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Rimessi P, Sabatelli P, Fabris M, Braghetta P, Bassi E, Spitali P, et al. Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse. Mol Ther. 2009;17:820–827. doi: 10.1038/mt.2009.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Sirsi SR, Schray RC, Wheatley MA, Lutz GJ. Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides. J Nanobiotechnology. 2009;7:1. doi: 10.1186/1477-3155-7-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Malerba A, Thorogood FC, Dickson G, Graham IR. Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice. Hum Gene Ther. 2009;20:955–965. doi: 10.1089/hum.2008.157. [DOI] [PubMed] [Google Scholar]
  • 51.Wu B, Li Y, Morcos PA, Doran TJ, Lu P, Lu QL. Octa-guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice. Mol Ther. 2009;17:864–871. doi: 10.1038/mt.2009.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Yin H, Moulton HM, Betts C, Seow Y, Boutilier J, Iverson PL, et al. A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice. Hum Mol Genet. 2009;18:4405–4414. doi: 10.1093/hmg/ddp395. [DOI] [PubMed] [Google Scholar]
  • 53.Ferlini A, Sabatelli P, Fabris M, Bassi E, Falzarano S, Vattemi G, et al. Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP-AON complexes. Gene Ther. 2010;17:432–438. doi: 10.1038/gt.2009.145. [DOI] [PubMed] [Google Scholar]
  • 54.Heemskerk H, de Winter C, van Kuik P, Heuvelmans N, Sabatelli P, Rimessi P, et al. Preclinical PK and PD studies on 2′-O-methyl-phosphorothioate RNA antisense oligonucleotides in the mdx mouse model. Mol Ther. 2010;18:1210–1217. doi: 10.1038/mt.2010.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Hu Y, Wu B, Zillmer A, Lu P, Benrashid E, Wang M, et al. Guanine analogues enhance antisense oligonucleotide-induced exon skipping in dystrophin gene in vitro and in vivo. Mol Ther. 2010;18:812–818. doi: 10.1038/mt.2009.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Wu B, Lu P, Benrashid E, Malik S, Ashar J, Doran TJ, et al. Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino. Gene Ther. 2010;17:132–140. doi: 10.1038/gt.2009.120. [DOI] [PubMed] [Google Scholar]
  • 57.Yin H, Betts C, Saleh AF, Ivanova GD, Lee H, Seow Y, et al. Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse. Mol Ther. 2010;18:819–827. doi: 10.1038/mt.2009.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Sharp PS, Bye-a-Jee H, Wells DJ. Physiological characterization of muscle strength with variable levels of dystrophin restoration in mdx mice following local antisense therapy. Mol Ther. 2011;19:165–171. doi: 10.1038/mt.2010.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Arechavala-Gomeza V, Graham IR, Popplewell LJ, Adams AM, Aartsma-Rus A, Kinali M, et al. Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle. Hum Gene Ther. 2007;18:798–810. doi: 10.1089/hum.2006.061. [DOI] [PubMed] [Google Scholar]
  • 60.Popplewell LJ, Adkin C, Arechavala-Gomeza V, Aartsma-Rus A, de Winter CL, Wilton SD, et al. Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials. Neuromuscul Disord. 2010;20:102–110. doi: 10.1016/j.nmd.2009.10.013. [DOI] [PubMed] [Google Scholar]
  • 61.Takeshima Y, Yagi M, Wada H, Ishibashi K, Nishiyama A, Kakumoto M, et al. Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy. Pediatr Res. 2006;59:690–694. doi: 10.1203/01.pdr.0000215047.51278.7c. [DOI] [PubMed] [Google Scholar]

Articles from Artificial DNA, PNA & XNA are provided here courtesy of Taylor & Francis

RESOURCES