Introduction

Anti-sense oligonucleotides (AOs) have been used to manipulate splicing of the dystrophin pre-messenger RNA to induce specific exon skipping in vitro and in vivo, in animal models of muscular dystrophy and in cells from Duchenne muscular dystrophy (DMD) patients. Disease-causing mutations in the dystrophin gene can be eliminated by removal of exons that contain non-sense mutations, or exons that flank frame-shifting deletions, to produce an in-frame transcript (for a review, see refs.^1,2,3,4). DMD is a systemic disease (for a review, see refs. 5,6), and for dystrophin exon skipping to be of benefit in ameliorating human disease, it will be necessary to achieve systemic distribution of the anti-sense agent.

The dystrophin gene is prone to deletion in two regions in particular,⁷ but all types of mutation have been described across the dystrophin gene, including deletions, duplications, inversions, non-sense mutations, and splicing errors.^8,9,10 The milder allelic disorder, Becker muscular dystrophy, is usually caused by in-frame deletions in the dystrophin gene.¹¹ The dystrophin transcripts of patients with this disorder^{12,13,14,15,16} and the transcripts responsible for the rare, dystrophin-positive revertant fibers in dystrophic muscle^{17,18,19,20,21} indicate potential exon combinations that may be most appropriate for ameliorating DMD. Transcript manipulation allows for tissue-specific dystrophin expression under endogenous regulation, and of all the currently proposed treatments to address DMD, selected exon exclusion is the only option that has natural precedents.

Oligonucleotide analogues administered to animal models of muscular dystrophy and evaluated in human myogenic cells have shown variable ability to remove the target exons.^22,23,24,25 We have previously reported that a phosphorodiamidate morpholino oligomer (PMO) appeared superior at excluding exon 23 from the dystrophin transcript in vitro²⁵ and in vivo^23,25 in the mdx mouse model of muscular dystrophy, compared with a 2'-O-methyl phosphorothioate AO of identical sequence. In this study, we report that enhanced PMO uptake, mediated by a cell-penetrating peptide (CPP) delivery vector,²⁶ results in systemic dystrophin expression and reduced dystrophic pathology in mdx mice at a significantly lower dose than used in other studies.^23,27 We propose that as a result of the enhanced uptake of the PMO conjugated to the CPP (PMO-pep), safety and efficacy can be optimized because of the lower dosages required for induced dystrophin expression.

Results

Dystrophin expression in mdx mouse tissues after PMO-pep treatment

Dose evaluation. Neonatal mdxmice, 1–2 days old, were injected intraperitoneally (ip) with a single dose of 1, 2, 5, 10, or 25 mg/kg PMO-pep in normal saline. The mice were killed 2 weeks later. Tissue sections were analyzed for dystrophin expression by immunofluorescence (Figure 1). Dystrophin expression in diaphragm increased with PMO-pep dosage, reaching levels comparable to those of normal C57BL/10ScSn mice after a single treatment of 10 mg/kg.

Figure 1.

Dystrophin expression in diaphragm from mdx mice 2 weeks after a single treatment with PMO-pep at dosages ranging from 1 to 25 mg/kg. Treatment commenced at 1–2 days of age. Dystrophin was detected with NCL-DYS2 and Zenon Alexa Fluor 488 on 6-m unfixed cryosections. Sections from age-matched sham-treated mdx and C57BL/10ScSn mice are included for comparison (bar = 200 m).

Full figure and legend (63K)

Long-term evaluation. In an attempt to develop a treatment regimen that would induce systemic dystrophin expression at the lowest possible dosage, three litters of mdx mice pups were given four once-weekly ip injections of 1, 2 (data not shown), or 5 mg/kg, beginning at 1 day of age, and analyzed for dystrophin expression by reverse-transcription–polymerase chain reaction (RT-PCR) at 6 weeks of age. The full-length transcript is represented by an amplicon of 901 base pairs, and the in-frame transcript excluding exon 23 is represented by the 688-base-pair product. Treatment with PMO-pep at a dosage of 1 mg/kg/week induced low levels of exon 23 skipping and dystrophin expression in diaphragm, as demonstrated by immunofluorescence; however, dystrophin expression was not detected in other tissues. Dystrophin levels were increased in diaphragm in mdxmice treated with 2 mg/kg/week PMO-pep, but only very low levels of expression were observed in distal muscles (data not shown). Figure 2a shows representative levels of native and induced dystrophin transcripts from an mdxmouse 3 weeks after the final treatment with 5 mg/kg PMO-pep. The amplicon excluding exon 23 (688 base pairs) appears as a major product in all tested tissues from treated mice, with the exception of cardiac muscle. The full-length transcript is present in all tissues except the PCR negative control and the diaphragm sample. In the latter case, exon skipping efficiency appeared to be 100%, as no full-length product was observed. No shortened transcript was detected in muscle from saline-injected mice. The 542-base-pair amplicon represents an out-of-frame transcript missing exons 22 and 23 and has been previously reported.^28,29

Figure 2.

Dystrophin expression in tissues from mdx mice treated with four once-weekly intraperitoneal injections of PMO-pep at 5 mg/kg, commencing at 1–2 days of age. (a) Reverse-transcription–polymerase chain reaction (RT-PCR) analysis of tissues from an mdxmouse at 6 weeks of age. The 901-base-pair (bp) product represents the full-length transcript, and the products of 688 and 542 bp represent transcripts that exclude exon 23 and exons 22 and 23, respectively. (b) Dystrophin detected in representative tissues from a treated mdx mouse, 6 weeks of age, by western blotting using NCL-DYS 2 and visualized by chemiluminescence. Sample loading was standardized according to myosin content as determined by densitometry. (c) Dystrophin expression in tissues from mice that received four once-weekly treatments of PMO-pep, commencing at 1–2 days of age, and analyzed at 6, 8, 12, and 26 weeks. Dystrophin was detected by staining with NCL-DYS2 and Zenon Alexa Fluor 488 on 6-m unfixed cryosections (D, diaphragm; Quad, quadriceps femoris; TA, tibialis anterior; GM, gluteus maximus; TB, triceps brachialis). Sections from sham-treated mdx and C57BL/10ScSn mice aged 8 weeks are included for comparison. Tri, trichome (bar = 200 m).

Full figure and legend (163K)

Western blotting on samples from treated mdx mice (Figure 2b) confirms that four once-weekly injections of PMO-pep at 5 mg/kg per treatment induced normal levels of dystrophin in diaphragm (110%) and levels of 8 and 3% in tibialis and gluteal muscles, respectively, as determined by densitometry and comparison with C57BL/10ScSn tibialis muscle, 6 weeks after the initial treatment commencing at 1 day of age (3 weeks after the final treatment). No dystrophin was detected in cardiac muscle or in saline-injected controls.

Persistence of the induced dystrophin expression was evaluated in mice at 6, 8, 12, and 26 weeks of age after four once-weekly treatments with 5 mg/kg PMO-pep and compared with results for sham-treated mdxand C57BL/10ScSn mice (8 weeks old). Dystrophin staining and muscle architecture appeared near-normal in diaphragm from treated mice aged 6, 8, and 12 weeks compared with C57BL/10ScSn mice (Figure 2c). Although some dystrophin was present in the diaphragm from the treated mice aged 26 weeks (i.e., 23 weeks after the final treatment at 3 weeks of age), the staining appeared discontinuous in some areas and muscle architecture shows some disruption. At 6 weeks of age, tissue samples from the treated animals show dystrophin expression and near-normal muscle architecture, except for the heart, which has no detectable dystrophin. Sections of quadriceps from these mice, labeled with NCL-DYS2 and Alexafluor 488, and Hoechst to visualize nuclei, demonstrated normalized pathology and central nucleation (data not shown).

In mice of 8 and 12 weeks of age, dystrophin expression in tibialis anterior is limited to small groups of fibers. Dystrophin expression in colon was maintained at 12 weeks, but it declined by 26 weeks.

To demonstrate that uptake of the PMO was not limited to neonatal mice, animals aged 1 day, 4 weeks, and 1 year were given four once-weekly treatments of PMO-pep at a dosage of 5 mg/kg and analyzed 8 weeks after the first treatment (i.e., at ages 8, 12, and 60 weeks). Dystrophin staining on representative diaphragm sections from all treated mice showed similar intensity to that seen in the C57BL/10ScSn sample (Figure 3a). No dystrophin was observed in the section from a sham-treated mdxmouse. The accumulation of scar tissue in mice aged 12 weeks and older is revealed by Picro Mallory trichrome staining (Figure 3b), and hematoxylin and eosin staining (Figure 3c) shows the marked infiltration of mononuclear cells. Treatment of neonatal mdx mice with PMO-pep (5 mg/kg/week) appears to abrogate the onset of the dystrophic pathology in the diaphragm. There is no evidence of infiltrating mononuclear cells, and connective tissue and muscle fibers appear normal. The same treatment regimen in older mice, commencing at 4 weeks and at 1 year of age, did not abolish the pre-treatment dystrophic pathology and disrupted muscle architecture in diaphragm, but it did result in substantial dystrophin expression (Figure 3a) and a marked reduction in mononuclear cells (Figure 3c). The functionality of the induced dystrophin is demonstrated by the uniformity of the muscle fibers in the hematoxylin and eosin–stained section of diaphragm in animals treated as a neonates.

Figure 3.

Dystrophin expression and morphology of diaphragm in mdx mice treated with PMO-pep at different ages. (a) Dystrophin expression in diaphragm from mice receiving four once-weekly treatments of PMO-pep at 5 mg/kg, commencing at 1 day, 4 weeks, and 52 weeks of age, analyzed at 8, 12, and 60 weeks of age. Dystrophin was detected by staining with NCL-DYS2 and Zenon Alexa Fluor 488 on 6-m unfixed cryosections. Diaphragm cryosections from the treated mdx mice (as above) and age-matched sham-treated mdx mice were stained with (b) Picro Mallory trichrome and (c) hematoxylin and eosin (H&E). Sections from C57BL/10ScSn mice aged 20 weeks are included for comparison (bar = 200 m).

Full figure and legend (167K)

Animal monitoring

Growth rates of treated mice were monitored and compared with those of the untreated mdxand C57BL/10ScSn mice (Figure 4). There was no apparent difference in growth rates between mice in the various groups, until the age at which mdx mice begin to show evidence of muscle degeneration and regeneration (14–18 days). From this time, mdx mice are smaller than C57BL10/ScSn mice (P = 0.007); however, they continued to gain weight as adults, when the C57BL10/ScSn mice showed slower weight gain, and by approximately 50 days of age, the mice were of similar size. Mice treated with four once-weekly doses of PMO-pep at 5 mg/kg, from 2 to 22 days of age, had a slightly lower mean weight than untreated mdxmice; however, this was not significant and the mice were of similar size at 50 days of age.

Figure 4.

C57BL/10ScSn (closed inverted triangle), n = 59; sham-treated mdx (open circle), n = 79; and mdx mice that received four once-weekly treatments of PMO-pep (5 mg/kg) at 2, 8, 15, and 21 days of age (closed circle), n = 11, were weighed between 2 and 52 days of age. The weights of treated mdxand C57BL/10ScSn mice were significantly different, as indicated (*P < 0.007), whereas those of treated and untreated mdx mice were not.

Full figure and legend (4K)

To monitor the effect of the treatment and the general health of the mice, liver transaminases, alkaline phosphatase, and creatinine were measured in serum samples from PMO-pep-treated and sham-treated mdx mice, and from C57BL10/ScSn mice aged 3–12 weeks. All parameters except alkaline phosphatase were elevated in mdxmice compared with C57BL10/ScSn mice; however, there was no significant difference between the PMO-pep-treated and sham-treated mdx mice (P > 0.05), indicating that the PMO- pep regimen at this dose is unlikely to be causing additional tissue damage (data not shown). In mdxmice aged 3–12 weeks that received four once-weekly injections of PMO-pep at 5 mg/kg per treatment, mean serum creatine kinase levels were approximately 50% of those of sham-treated age-matched controls; however, these levels were higher than those in C57BL10/ScSn mice (data not shown).

Discussion

A number of approaches, including gene or cell replacement, homologous gene up-regulation, and non-sense mutation suppression are being investigated to address the dystrophin deficiency in DMD (for a review, see refs. 4, 30). To date, the most significant improvements in DMD prognosis have come from steroid treatment (for a review, see refs. 31, 32) and nocturnal mechanically assisted ventilation.³³ It is not unreasonable to assume that if reduced respiratory stress can substantially improve the life of a DMD patient, benefits from any treatment that partially restores dystrophin expression are likely to be significant, particularly when used in conjunction with the current management practices.

Targeted exon skipping will not be applicable to all DMD patients, in particular those affected by large genomic deletions or the loss of essential coding domains. The efficient application of specific exon skipping to bypass dystrophin mutations could result in a shortened dystrophin of at least partial function, and it has been estimated that up to 75% of DMD patients could benefit from exon removal to overcome dystrophin mutations.³⁴ Although genomic deletions including large or essential protein-coding domains may be amenable to AO-mediated messenger RNA reading frame restoration, the resultant protein could be functionally compromised.

The utility of AO-induced exon skipping in restoring dystrophin expression in experimental systems has been demonstrated.^{23,25,28,29,35,36,37} The current challenge is to achieve sustained dystrophin expression accompanied by minimal adverse effects in most, if not all, tissues affected by the absence of dystrophin. Ideally, anti-sense compounds for induced exon skipping should be directed to the most amenable target motif, show efficacy at low doses, have sustained action, and be easily delivered as a simple formulation.

Previously, we evaluated different anti-sense chemistries in vivo and observed that PMOs induced substantial shortened dystrophin transcript 2 weeks after a single intramuscular injection.²³ It is generally assumed that if AO-induced exon skipping is to provide the greatest benefit to DMD patients, it will be necessary to commence treatment before irreversible muscle loss has occurred. Although intramuscular injection of AOs is useful in evaluating the efficacy of compounds in vivo, any treatment for DMD will need to be effective after systemic delivery. Because skeletal muscle constitutes approximately 30% of the total body mass and because dystrophin isoforms are also expressed in a variety of tissues, DMD can be addressed only by systemic dystrophin restoration.

PMO anti-sense compounds have been shown to be effective in removing target exons from the dystrophin pre-messenger RNA,^23,25,27 are reputedly non-toxic,³⁸ and have minimal non-anti-sense effects.^39,40 We now report that systemic PMO distribution, efficient intracellular uptake of the oligomer, and targeted exon exclusion are greatly enhanced by using a CPP conjugated to the PMO. Dystrophin expression in diaphragm and limb muscles was demonstrated using RT-PCR, western blotting, and immunofluorescence.

Normalized dystrophin expression in diaphragm and marked expression in smooth muscle of the gut after ip delivery of the PMO-pep could have been mediated, in part, by diffusion from the abdominal cavity. However, exon 23 skipping and uniform dystrophin expression in distal muscles, albeit at lower levels, can have resulted only from systemic distribution of the oligomer. The dosages of PMO-pep (125 g/adult mouse, once weekly) are markedly lower than those used previously for an unconjugated PMO (625 g/adult mouse, thrice weekly).²³ Using a substantially higher dosage, Alter et al.²⁷ reported dystrophin expression after seven intravenous injections of unconjugated PMO at a dosage of 2,000 g/mouse once weekly in adult mice. At the dosages used in our current report, the peptide vector and PMO do not appear to cause any obvious adverse effects that could be detected by routine observation and blood biochemistry analysis. Serum transaminases are normally elevated when muscle damage is present, and we show that there was no further elevation of these parameters in mice treated with PMO-pep, indicating that the administration of the compound at the dosages used here was unlikely to have had adverse effects on the liver.

Use of the ip delivery route permits treatment of neonatal animals and allowed us to demonstrate that PMO-induced dystrophin can largely prevent onset of the dystrophic process that normally begins at approximately 14 days of age.⁴¹ Treatment of older mice restored dystrophin expression in diaphragm and reduced the number of mononuclear cells invading the tissue; however, as expected, the dystrophic pathology was not reversed, as the muscle damage and dystrophic phenotype were already well established.

The progression of muscle wasting in DMD is relentless, and all muscle is affected. It would be preferable for induced exon skipping treatment to commence as early as possible, before the muscle architecture is severely disrupted. We have shown that dystrophin expression can persist in the mdxdiaphragm for at least 23 weeks after treatment. This may be due to the fact that dystrophin expression in this muscle was essentially normalized in the young adult mouse, and, thus, the dystrophin-associated complex would be stable and the muscle would not have been susceptible to damage. Distal muscles expressing lower levels of dystrophin would probably have been less protected, and thus subject to some turnover. The absence of induced exon skipping and detectable dystrophin in cardiac muscle after PMO treatment is a concern that has been reported by others²⁷ and may reflect tissue-specific differences in oligomer uptake or splicing mechanics, or both. Additional long-term studies are required to refine the treatment regimen and to determine the level, extent, and duration of dystrophin expression that is necessary to prevent muscle loss and preserve muscle function at a dose that has minimal adverse effects.

Amelioration of the dystrophic phenotype by exon skipping has been reported only in the mdx mouse;^36,37 however, dystrophin exon skipping in human cells has been reported.^22,35,42,43 It is yet to be demonstrated that removal of dystrophin exons can reduce the phenotype severity in DMD or Becker muscular dystrophy patients. Although it is predicted that many DMD patients could benefit from AO-induced exon skipping, each mutation will need to be evaluated independently. It is probable that benefits from the treatment will vary substantially among patients, and responses are likely to be dependent upon the nature and position of the mutations as well as the age at which treatment commences.

A major shortcoming of exon removal to treat DMD is that the anti-sense compounds will need to be administered periodically first to establish and then to maintain dystrophin expression. In an attempt to overcome this limitation, persistent dystrophin exon 23 skipping in the mdx mouse has been achieved by the single administration of an adeno-associated virus vector expressing anti-sense sequences linked to a modified U7 small nuclear RNA.⁴⁴ However, the requirement for periodic re-administration and the long-term safety of this treatment are yet to be determined. Because the viability of repeated use of viral vectors in the clinic remains to be established, an anti-sense compound that has extended biological stability, minimal toxicity, and non-anti-sense effects would be highly desirable. The stability of the morpholino structural type²⁶ and its favorable safety profile,³⁸ together with the data presented here and reported previously,^23,27 provide compelling reasons to explore PMO compounds further for splicing manipulation to overcome the human disease.

Materials and Methods

Anti-sense oligomer. The PMO, designated M23D(+7–18), anneals to the last 7 nucleotides of mouse dystrophin exon 23 and the first 18 nucleotides of intron 23 and has been described previously.^23,25 The PMO, prepared as a conjugate with the CPP, was supplied by AVI Biopharma (Corvallis, OR) and is referred to here as PMO-pep. The CPP had the sequence of (RXR)₄XB (where R is arginine, X is 6-aminohexanoic acid, and B is -alanine). Non- amino acid X was incorporated into the octa-arginine R₈ sequence to increase the peptide's stability in blood circulation.²⁶ A PMO-pep conjugate was found to be bound primarily to cell membrane proteoglycans and internalized by an endocytosis mechanism.⁴⁵ By an unknown mechanism, a fraction of endocytosed conjugate was released from endosomal/lysosomal compartments to reach nuclei,⁴⁵ correcting pre-messenger RNA mis-splicing⁴⁵ and inducing exon skipping in an in vitro DMD model.⁴⁶ A PMO conjugated to (RXR)₄XB was found to be much more efficient at escaping these compartments than the PMO conjugated to other well-known CPPs such as R₉ and Tat peptides.⁴⁵

Animals. Mice were supplied by the Animal Resources Centre, Murdoch, Western Australia, and housed according to National Health and Medical Research Council (Australia) guidelines. The use of animals was approved by the Animal Ethics Committee of the University of Western Australia (approval number 03/100/373). C57BL/10ScSn^mdx mice (mdx)⁴⁷ carry a non-sense mutation in exon 23 of the dystrophin gene.⁴⁸

Systemic delivery of PMOs. The PMO-pep was re-suspended in sterile, purified water at a concentration of 5 mmol/l (51.69 mg/ml) and stored at 4 °C until required. The PMO-pep diluted in normal saline was delivered to neonatal and adult mdx mice by the ip route, at dosages of 1–25 mg/kg per treatment. Sham-treated animals were injected with an equivalent volume of normal saline. In longer-term experiments, the PMO-pep was injected into neonatal mdx mice, by the ip route, beginning 1–2 days after birth and repeated weekly until the mice were 3 weeks old (four injections in total). Mice were weighed daily to detect any adverse effects on growth. Mice were anaesthetized by Rhodia Halothane (Merial, Sydney, Australia) inhalation and then killed by cervical dislocation. Tissues were dissected and frozen in isopentane, cooled by liquid nitrogen, before analysis by RT-PCR, Western blotting, and immunofluorescence.

RNA preparation and RT-PCR analysis. Total RNA was extracted from 2–3 mg of sections, cut from frozen tissue blocks, using Trizol (Invitrogen, Melbourne, Australia) according to the manufacturer's protocol. RT-PCR was performed across dystrophin exons 20–26 and evaluated as previously described.^28,29

Protein extraction and western blotting. Protein extracts were prepared for Western blotting as described previously.²³ Dystrophin was visualized using NCL-DYS2 monoclonal anti-dystrophin (Novacastra, Newcastle-upon-Tyne, UK) at a dilution of 1:100 for 2 hours at room temperature, with subsequent detection using the Western Breeze protein detection kit (Invitrogen, Melbourne, Australia). Images were captured directly by a Chemi-Smart 3000 gel documentation system using Chemi-Capt software for image acquisition and Bio-1D software for image analysis (Vilber Lourmat, Marne-la-Vallée, France).

Dystrophin immunofluorescence. Dystrophin was detected in 6-m unfixed cryostat sections using the Novacastra NCL-DYS2 monoclonal antibody, which reacts strongly with the C-terminus of dystrophin. Immunofluorescence was performed using the Zenon Alexa Fluor 488 labeling kit (Invitrogen, Melbourne, Australia), according to the protocol recommended by the manufacturer, but omitting the initial fixation step. The primary antibody was used at a dilution of 1:10 with a molar ratio of 4.5:1. Additional sections were stained with Picro Mallory trichrome to visualize connective tissue or hematoxylin and eosin to assess the muscle architecture. Sections were viewed with an Olympus IX 70 inverted microscope and the images captured on an Olympus DP 70 digital camera (Olympus Australia Pty Ltd, Sydney, Australia). Images in each montage were photographed using the same parameters to allow comparison.

Clinical biochemistry. Analysis of serum creatine kinase, liver enzymes, and creatinine was performed by the Murdoch University Veterinary Hospital School of Veterinary and Biomedical Sciences.

References

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Acknowledgments

The authors received funding from the National Institutes of Health (RO1 NS044146-02), the Muscular Dystrophy Association USA (MDA3718), the National Health and Medical Research Council of Australia (303216), and the Medical and Health Research Infrastructure Fund of Western Australia. P.L.I. and H.M.M., who supplied the PMO-pep, disclose that they hold stock in a company that has an interest in developing therapeutic anti-sense oligomers. This may be perceived as a conflict of interest.