Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice

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Abstract

Antisense therapy with both chemistries of phosphorodiamidate morpholino oligomers (PMOs) and 2′-O-methyl phosphorothioate has demonstrated the capability to induce dystrophin expression in Duchenne muscular dystrophy (DMD) patients in phase II-III clinical trials with benefit in muscle functions. However, potential of the therapy for DMD at different stages of the disease progression is not understood. In this study, we examined the effect of peptide-conjugated PMO (PPMO)-mediated exon skipping on disease progression of utrophin-dystrophin-deficient mice (dko) of four age groups (21–29, 30–39, 40–49 and 50+ days), representing diseases from early stage to advanced stage with severe kyphosis. Biweekly intravenous (i.v.) administration of the PPMO restored the dystrophin expression in nearly 100% skeletal muscle fibers in all age groups. This was associated with the restoration of dystrophin-associated proteins including functional glycosylated dystroglycan and neuronal nitric synthase. However, therapeutic outcomes clearly depended on severity of the disease at the time the treatment started. The PPMO treatment alleviated the disease pathology and significantly prolonged the life span of the mice receiving treatment at younger age with mild phenotype. However, restoration of high levels of dystrophin expression failed to prevent disease progression to the mice receiving treatment when disease was already at advanced stage. The results could be critical for design of clinical trials with antisense therapy to DMD.

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by out-frame and nonsense mutations in the dystrophin gene. DMD patients show their first symptoms of muscle weakness commonly around 3 years old. The disease progresses rapidly and patients are usually confined to a wheel chair in their early teens.1 Patients often die by their early twenties due to cardiac and respiratory failure, although improved patient care has now significantly extended their life span. However, there is still no effective treatment for the disease. Recently, antisense oligonucleotide-mediated exon skipping has emerged as a highly promising experimental therapy.2 Preclinical studies have provided concrete evidence that exon skipping is an effective strategy for the restoration of dystrophin expression in cell culture and in animal models with DMD mutations.3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 Long-term regular administration of antisense oligonucleotides in mdx mice was able to achieve high levels of skipping of the mutated exon 23 and maintain levels of dystrophin expression in body-wide muscles with improvement in muscle function and pathology.22,23 Phase I and II clinical trials with two chemistries, 2′-O-methyl phosphorothioate and phosphorodiamidate morpholino oligomers (PMOs) as antisense oligomer drugs targeting dystrophin exon 51, have validated the preclinical conclusion that selected antisense oligomer sequences of the two chemistries can achieve targeted exon skipping and restoration of dystrophin expression in DMD patients.24, 25, 26, 27, 28 More recently, extended phase IIb clinical trials reported a noticeable delay in disease progression for DMD patients receiving weekly i.v. injection of 30–50 mg kg−1 PMO drug targeting exon 51 for more than 1 year.28 However, GlaxoSmithKline and Prosensa announced that its phase III clinical trial (NCT01254019) of Drisapersen (6 mg kg−1 weekly injection) failed to meet the primary end point of a statistically significant improvement in the 6-min Walking Distance Test compared with placebo (http://www.gsk.com/media/press-releases/2013/gsk-and-prosensa-announce-primary-endpoint-not-met-in-phase-iii-.html). This raises the question whether the current treatment regime for both chemistries is sufficient to achieve long-term clinical significance to the DMD patients. The severity of the DMD is determined by the progressive loss of muscle fibers as a result of degeneration and limited capacity of human muscles to regenerate. Therefore, therapeutic value of any therapy which is unable to increase the regeneration capacity will depend on the amount of remaining muscles within individual DMD patient. Exon skipping therapy rescues muscle function by restoring dystrophin expression in existing muscle fibers, but will not be able to restore the lost muscle fibers. This implies that the therapeutic significance of exon skipping may depend on the stage of disease progression and the time of intervention. For example, patients already wheelchair bound with depleted muscle mass and severe fibrosis might benefit little from exon skipping therapy. However, experiments to correlate disease severity to clinic outcome with exon skipping therapy have not been well documented. Almost all previous exon skipping studies in vivo have used the mdx mice as the model. However, mdx mice apparently have a high capacity to regenerate and are able to maintain skeletal muscle mass comparable to normal mice, although increase in fibrotic tissue and fat deposition become obvious at the old age.29 Also important, the mdx mice can live up to 2 years of age, which is comparable to the life span of wild-type mice, making it difficult to assess the potential effect of the therapy on longevity, one most reliable and important marker for benefit assessment. Previous study used a more severe dystrophic dog model to evaluate the efficacy of exon skipping and demonstrated functional improvement by restoration of reading frame with PMO antisense oligomers.19 However, the study was unable to determine the effect of the therapy on life span, because of limited number of dogs available.

Knockout of utrophin, a paralog of dystrophin and normally expressed at neuromuscular junctions, in mdx mice background (utrophin-dystrophin-deficient mice––dko) produces much severer disease phenotype than mdx mice.30 The dko mice show many signs typical of human DMD and progressive muscle weakness that lead to premature death largely within 15 weeks. The physical characteristic of weight loss soon after weaning, early onset of joint contractures and kyphosis together with short life span are highly valuable markers for assessment of therapeutic intervention. The dko mice are therefore considered pathologically more relevant to DMD in human especially for testing therapeutic potential as an in vivo model system.

In this study, we examined the therapeutic value of the exon skipping strategy in the dko mice by their physical characteristics in combination with evaluation of pathology and life span. The results show that the degree of protection provided by PMO-mediated exon skipping against disease progression is closely related to the timing of intervention. Treatment given in the early stage (between 20 and 29 days) of the disease effectively prevents the development of severe joint contracture and kyphosis. Almost all treated mice maintained a steady weight increase and lived healthy lives for more than 6 months until the termination of the study. Despite similar efficiency in exon skipping and dystrophin expression, delayed administration of PPMO severely reduces the efficacy of the treatment. Treatment of PPMO could not effectively reverse the phenotype of dko mice already with severe joint contracture, kyphosis and weight loss and was unable to prolong life span of the animals. These results are valuable for design and evaluation of clinical trial with exon skipping therapy.

Results

Early PPMOE23 treatment rescues phenotype and significantly extends life span of the dko mice

Our previous studies have shown that monthly injections of 30 mg kg−1 PPMOE23 for 1 year improved the levels of serum creatine kinase and histology to near normal with significantly enhanced muscle functions in mdx mice. Considering the potential acute toxicity of peptide-conjugated PMO (PPMO), we applied a 15 mg kg−1 PPMOE23 biweekly i.v. injection regime to treat the dko mice. Four different age groups of dko mice (20–29, 30–39, 40–49, 50+ days old, referred as 20–29D to 50+D) received treatment with the PPMOE23 targeting mouse dystrophin exon 23 reported previously.13,21

Untreated dko mice have a relatively mild disease phenotype before the age of 28 days, although dystrophic pathology indicated by central nucleation and degenerating fibers was readily detected as previously reported.28 When held by the tail there was no observable gait posture or leg muscle contracture indicated by retraction of the hindlimb. Also, there was no clear kyphosis noted. Body weight of untreated dko mice increased steadily from birth reaching 15 g in average by 28 days, although the weight was smaller than littermates of utrophin+/−/dystrophin mice. However, the disease progress accelerated afterward with development of clear muscle contracture and kyphosis.

Body weight of the dko mice started to decline after 50 days and eye infection appeared at the similar time and persisted in 70% of the mutant mice. Rapid muscle wasting and severe kyphosis led to difficulty in breathing. More than half of the dko mice died within 70 days and the rest of the mice all died within 90 days in our condition (Figure 1).

Figure 1
figure1

Effect of PPMOE23 treatment on disease phenotype and life span of the dko mice. Untreated, control dko group. Numbers represent the age group of dko mice when the treatment starts. (a) Life spans of dko mice from untreated and treated groups. (b) Mice body weight changes during the treatment from untreated and treated groups. (c) Mice body size comparison, appearance and abnormal limb contraction of 70-day-old mice from untreated, 20–29D and 40–49D treated groups. (d) Onset of abnormal walking limb posture from untreated and treated groups. (e) Percentages of mice with eye infections from untreated and treated groups. (f) Kyphosis detected by X-ray images from untreated and treated groups. (g) Kyphosis index analysis from untreated and treated groups. (h) Onset of kyphosis from untreated and treated groups. Mean±s.e.m., n=10. Statistical differences between treatment groups and control groups were evaluated by Student’s t-test. *P<0.05.

Treatment with the PPMOE23 (15 mg kg−1 biweekly) starting at the age of 20–29 days greatly delayed the development and deterioration of gait posture and kyphosis in most mice (Figure 1c). Only 2 of the treated mice in this group (total 10) started showing muscle weakness with visible gait posture and mild kyphosis starting from 92 and 125 days. All other mice of the group were still able to stand on their hind legs and reaching out of the cage with near normal walking limb posture during the entire period of the treatment up to the time of termination. No eye infection was observed in any mouse (Figures 1c, d and f). Consistently, body weight of the treated mice continued to increase steadily reaching 28 g in average. However, one treated mouse with kyphosis visible at 92 days deteriorated afterwards and body weight declined rapidly (Figure 1b). The mouse further developed difficulty in breathing and movement and was terminated by humane end point at the age of 161 days. All other mice including the one with visible kyphosis from 125 days maintained normal food consumption and activity up to the termination date. Postmortem X-ray examination showed that the surviving mice presented only mild kyphosis, whereas the one died at 161 days showed severe kyphosis (Figure 1a).

Dystrophic phenotype with mild gait and muscle contracture became apparent in the majority of the untreated dko mice during the period of 30–39 days. The same PPMO treatment regime achieved less favorable outcome to this group mice than to the 20–29D group mice. Progression of spinal cord deformation, joint contractures and wadding gait were significantly delayed, but the signs of kyphosis gradually worsened around 83–113 days of age in most mice. By this age, however, most treated mice maintained ability to stand on their hind legs (Figure 1d). Only one mouse suffered persistent eye infection (Figure 1e). Body weight of the treated mice continued to increase, but measured at about 3 g less than those of the 20–29D group throughout the treatment period, reaching 25 g in average by the time of termination (180 days; Figure 1b). However, two mice with visible kyphosis from 47 days deteriorated progressively with breathing difficulty and movement limitation and terminated at 150 and 168 days. Another mouse with mild kyphosis at the beginning of the treatment, however, maintained the phenotype with slow disease progression and survived to 180 days, although kyphosis became very obvious. All of other seven mice survived with mild to moderate kyphosis at the time of study termination (Figures 1f–h).

Delayed PPMOE23 treatment has limited effect on disease phenotype and disease progression

The health of the untreated mice started to deteriorate more rapidly from the age of about 40 days with all mice exhibiting gait posture and kyphosis. PPMO treatment starting between 40 and 49 days failed to prevent disease progression effectively. Gait posture and kyphosis continued to deteriorate despite the PPMOE23 treatment (Figures 1c and d). Three mice suffered persistent eye infection and terminated at humane end point between 44 and 82 days (Figure 1e). However, despite the presence of severe skeletal deformation, four mice survived up to 180 days (Figure 1a). The body weight of these four mice increased slightly from the time the treatment was given to the end point (Figure 1b). The severity of kyphosis measured by the postmortem X-ray analysis indicated a slight improvement at the humane end point when compared with the untreated controls (Figures 1f–h).

No significant improvement was observed in the severity of kyphosis for the mice receiving the same treatment at the age 50 days or above (Figures 1f–h). Eye infection persisted in more than 40% of the mice and body weight continued to decline as observed in untreated controls (Figure 1b). Similar to the controls, all the mice had to be humanely terminated within the age of 70 days in average. All mice of the 50+D group received only one or two times of PPMO treatment before termination (Figure 1a).

PPMOE23 treatment restores near normal levels of dystrophin expression in mice of all age groups

Our previous studies have demonstrated that single injection and monthly repeated injections of 30 mg kg−1 PPMOE23 restored dystrophin to more than 50% normal levels in all muscles including cardiac muscle of the mdx mice.13,23 In this study, the expression of dystrophin was examined 2 weeks after the final injection of the PPMOE23. Individual treated mouse was also examined at the time point by which a humane end point was concluded by the veterinarian. As expected, immunohistochemistry detected dystrophin expression in near 100% of fibers in skeletal muscles, including tibialis anterior (TA), quadriceps, biceps, gastrocnemius from all PPMO-treated dko mice of all age groups (Supplementary Figure 1). Similarly, dystrophin expression was detected in the majority of fibers of the diaphragm in all groups of treated mice. Dystrophin expression in the heart was considerable weaker than in skeletal muscles with only about 60% myocytes showing clearly visible signal. Variation in signal intensity was also clearly noticeable between different mice within the same age groups and between different age groups. The percentage of dystrophin-positive fibers in the heart, diaphragm and other skeletal muscles was not significantly different between the mice treated at younger ages (20–29D and 30–39D) and the mice treated at older ages (40–49D and 50+D). Nevertheless, signal for dystrophin in the 50+D group mice was less intensive when compared with that in the groups with treatment starting at the age of 20–39 days. This is likely due to the fact that half of the 50+D mice only received single dose of PPMO injection, whereas the other half of mice received twice of PPMOE23 (Figure 2a).

Figure 2
figure2

Restoration of dystrophin expression in all groups of dko mice treated with 15 mg kg−1 PPMOE23 biweekly. (a) Dystrophin expression in skeletal muscles and the heart in all treated groups. (b) Western blotting demonstrates the levels of dystrophin expression in the TA (tibialis anterior) muscle, diaphragm and the heart. Three samples for each treated group. Dystrophin detection is shown in the upper three lanes, and α-actin is used as a loading control from TA muscles. The low panel shows the quantification of dystrophin levels from TA, diaphragm and the heart of the treated groups with corresponding muscles of C57 mice as 100% (labeled as 1). (c) Detection of dystrophin exon 23 skipping by RT-PCR. E22-E23-E24 and E22-E24 representing normal mRNA and the mRNA with exon 23 skipped, respectively.

Consistent to the immunohistochemistry, the amount of dystrophin protein measured by western blots in the TA muscle of the three treated younger groups was above 70% of the levels of the normal control muscle. The amount of dystrophin in the diaphragm was lower, ranging from 54 to 70% of normal levels in mice of the three age groups. The levels of dystrophin expression were markedly lower in the heart than in other muscles, with only 10–20% of normal levels. Again, no significant difference was detected between the three younger groups of mice receiving PPMOE23 treatment (Figure 2b). The level of dystrophin expression in the cardiac and skeletal muscles of the 50+D group animals receiving only once or twice injections of the PPMO was lower than that detected in the same muscle of the other three age groups, but the difference was not statistically significant (Figure 2b).

Reverser transcription PCR (RT-PCR) results showed that all muscles from the PPMOE23-treated animals contained the spliced form of dystrophin mRNA with exon 23 skipped as the major specie. The intensity of the truncated mRNA varied between and within different age groups, but the percentage of the truncated dystrophin was above 50% of unskipped dystrophin mRNA in all muscles of all age groups (Figure 2c).

Expression of dystrophin-associated proteins after PPMOE23 treatment

To assess potential variation in expression of dystrophin-related proteins in different age groups, we compared the expression of neuronal nitric synthase (nNOS), functional glycosylated α-dystroglycan and β-integrin in the TA muscles of PPMOE23-treated and untreated control dko mice. As reported previously in the control dko mice, expression of functional glycosylated α-dystroglycan detected by immunohistochemistry decreased, whereas β-integrin expression increased. Restoration of dystrophin in the treated mice restored the levels of functional glycosylated α-dystroglycan and β-integrin closer to the levels detected in the muscles of wild-type C57 mice (Figure 3,Supplementary Figure 2). Expression of nNOS was also restored to near normal levels in the groups of mice receiving treatment before the age of 50 days (Figure 3). However, nNOS levels in the muscles of mice treated after age 50 days were considerably lower than that detected in the other three treated groups. As this group of mice received only one and two times PPMO treatment, one possible explanation is therefore that restoration of normal levels of nNOS may depend on relatively longer period of stable dystrophin expression and improvement of biophysical condition of the muscles.

Figure 3
figure3

Immunohistochemistry for membrane nNOS (with rabbit-anti-nNOS, Millipore), functionally glycosylated α-dystroglycan (with IIH6, Millipore) and β1-intergin (with goat-anti-β1 R&D system) expression after PPMOE23 treatment in tibialis anterior muscles of dko mice. Untreated, control group dko mice. Numbers represent the age group of dko mice when the treatment starts.

Histological and functional assessment

Skeletal muscles in the dko mice treated with PPMOE23 starting between 20 and 39 days of age presented a mild dystrophic pathology when examined at the termination of the study. The muscles contained relatively homogenous population of fibers, with less than 15% of fibers being smaller than 30 μm in diameters. These small fibers are indicator of muscle regeneration as the result of degeneration. Nearly all the muscle fibers remained centrally nucleated, suggesting the persistence of degeneration and regeneration in the muscle tissues despite high levels of dystrophin expression (Supplementary Figure 3). However, degenerating fibers were only occasionally detected, and no large areas of inflammation were observed. Similarly, collagen deposition between fibers was sparse in the muscles of treated animals, and no clear difference was detected from the muscles of normal C57 mice (Figures 4a and b). The most significant improvement was observed in the diaphragm of the dko mice treated from the young groups. The muscles of the 20–29D group mice showed regular spaced and highly uniform fibers with limited fibrotic tissue and only sparsely distributed infiltration. Diaphragm of the 30–39D group mice presented a similar histological feature to the 20–29D group mice, but with an increase in fibrotic tissue shown by collagen staining. However, wider areas of fibrosis and patchy infiltrates became obvious in the diaphragm of the 40–49D group mice when examined at the same termination point of 180 days. Diaphragms in the group 50+D mice exhibited similar pathology to those untreated dko, with prominent variation in fiber size, large areas of fibrosis and infiltration when terminated by humane end points (less than 70 days). Fibrosis and fat deposition were also commonly observed in the untreated controls and the group 50+D mice, but not seen in mice treated at younger ages. The apparent decrease in the amount of collagen deposition in the diaphragm from the untreated dko, treated 50+D group to 30–39D group and 20–29D group mice was demonstrated by Masson’s trichrome staining and illustrated in Figure 4b.

Figure 4
figure4

Effect of PPMOE23 treatment on muscle pathology and serum components. (a) Hematoxylin and eosin staining for muscles, the kidney and the liver. No pathologic change of the liver and the kidney was observed in the PPMO-treated groups. Numbers represent the age group of dko mice when the treatment starts. TA, tibialis anterior muscle. (b) Collagen deposition in diaphragms detected by Masson’s trichrome staining. The degree of collagen deposition is directly related to the delayed treatment. (c) Serum tests. Creatine kinase levels are most significantly reduced in the PPMOE23-treated 20–29D group dko mice, but not different in 50+D group when compared with the untreated group. Similar improvement in reduction of total bilirubin, ALT, ALP is also observed in the three younger treated groups. Mean±s.e.m., n=10. Statistical differences between treatment groups and control groups were evaluated by Student’s t-test. ALP, alkaline phosphatase; ALT, alanine transaminase.

Cardiac muscle degeneration with large areas of scar formation (fibrosis) was commonly seen in the untreated dko mice. Areas of fibrotic tissue and infiltration were also detected in the cardiac muscle of all the PPMO-treated dko mice including those mice terminated by the end of 6 months. However, the size of the fibrotic areas was smaller with less frequency in 20–29D and 30–39D treated group mice terminated at 6 months of age than those in untreated and 50+D treated groups. Mice receiving the PPMOE23 between the ages of 40 and 49 days exhibited fibrosis and infiltration similar to the 50+ group (Figure 4a).

Consistently, serum creatine kinase levels were significantly reduced in the 20–29D, and 30–39D groups, although the levels were still significantly higher than that in normal C57 mice. Significant reduction in serum creatine kinase was also detected in the 40–49D group when compared with the untreated control dko. However, creatine kinase levels in the 50+D group remained high, without significant difference to those of the untreated control dko mice (Figure 4c). Similarly, levels of alanine transaminase, alkaline phosphatase and total bilirubin were elevated in the untreated mice and mice treated at 40 days or above, but significantly reduced in mice treated at the age younger than 40 days (Figure 4c ). No pathologic change of the liver and kidney was observed by hematoxylin and eosin staining in all PPMOE23-treated mice (Figure 4a).

Discussion

It is generally recognized that human muscles have limited regeneration capacity, an important contributing factor for the progressive loss of muscle fibers in the muscular dystrophies especially in DMD. This raises the concern for the efficacy of gene replacement strategies, including gene therapy and exon skipping, to those patients who have already lost most of their muscle mass and mobility. Specifically, therapeutic value of exon skipping might well depend on the timing when the treatment is applied during the course of disease, possibly with diminishing efficacy as disease progresses. This hypothesis is now strongly indicated from the results of the current study in the dko mice, which present severe dystrophic phenotype and greatly shortened life span, highly relevant to DMD in clinics. Despite similar levels of dystrophin induction by the PPMOE23 in all muscles examined, earlier treatment with the exon skipping significantly delayed the disease progression, prevented severe kyphosis, eye infection and prolonged the life span. The eye infection in the dko mice is likely related to the clear dystrophic pathology in the extraocular muscles, which are spared in the mdx.31 However, these benefits diminished as the treatment was given later in the disease progression. Treating the dko mice with severe kyphosis and weight loss conferred very limited improvement in disease manifestation and life span. These results clearly suggest that earlier treatment is critical for exon skipping to achieve higher efficacy.

It is not clearly understood the reason(s) for the failure of high levels of dystrophin induction in both cardiac and skeletal muscle to alleviate disease progression significantly in later stage. One of the most likely explanations is the status of skeletal muscle, especially the diaphragm of the dko mice. The diaphragm has already experienced severe depletion of muscle fibers, which was replaced by fibrotic tissue from the age of 40 days, particularly 50 days and older. This together with severe kyphosis undermines the respiratory function causing difficulty in breathing in later stage life of dko mouse. Perhaps as expected, the depleted muscle mass with disorganized fiber arrangement and scar tissue could not be corrected by exon skipping therapy. Also important is the inability of the therapy to correct existing scar tissues in cardiac muscles as fibrosis persists in all PPMO-treated groups, although the areas of such fibrotic tissues are smaller in the mice treated earlier compared with the same aged mice treated later.

It is interesting to see how relevant the current data from the dko mouse can be to DMD patients in clinical trial and evaluation of exon skipping. Currently, clinical trials targeting exon 51 with PMO and 2′-O-methyl phosphorothioate both recruited patients with mobility. Interestingly, Sarepta recently reported in its press release that 30 and 50 mg weekly i.v. injections had significant effect in 6-min walk test for most boys. However, despite the same treatment regime, two patients in the 30 mg kg−1 cohort showed rapidly progressive decline in 6-min walk test. This led to the termination of the patients to receive further treatment.28 Although the exact reason for the rapid decline in disease progression of the two boys during the trial period is not explained, it is apparent that the same treatment regime with the ability to stabilize the disease in some DMD patients failed to prevent the rapid deterioration in others. The levels of dystrophin induction could have important roles for determining the degree of protection provided by the exon skipping treatment. Variation in the levels of dystrophin expression in different muscles and in different animals was observed in animal models and is also expected in different patients. However, low efficiency in dystrophin induction is unlikely to be the main cause as induction of dystrophin in muscles was confirmed in all participating patients. We therefore attempt to postulate that these two cases in clinical trials might represent the similar phenomenon we observed in the dko mice. Induction of dystrophin by exon skipping might also have limited therapeutic effect when DMD has entered or is entering stages of rapid progression. This will become clearer when more clinical trial data become available.

Reduction in the percentage of centrally nucleated muscle fibers can be detected as early as 1 month in dystrophic muscles expressing high levels of dystrophin with PMO-mediated exon skipping in mdx mice. Significant reduction in central nucleation has been achieved in muscle treated with PMO and PPMO for longer than 3 months.16, 17, 18, 19, 20, 21, 22, 23 However, central nucleation persists in nearly all fibers of the skeletal muscles in the dko mice after more than 5 months of PPMO treatment, even in the group receiving treatment at the ages between 20 and 29 days. This result appears also contradictory to the report with adeno-associated virus-mediated micro-dystrophin expression in the dko mice.32 More than 50% reduction in centrally nucleated fibers was reported 1 year after the delivery of dystrophin transgene to the mice of 1 month old. It is unlikely that the dystrophin with exon 23 skipped will have less function than the microdystrophin expressed by the viral vector. However, it is possible that the levels of microdystrophin are significantly higher than that induced by exon skipping, thus providing better protection. Nevertheless, lack of utrophin expression, which is important for muscle differentiation as reported previously, likely also has important role for the lower efficiency in reducing the rate of central nucleation.33

In summary, our results provide the first and clear evidence that therapeutic value of dystrophin restoration by exon skipping depends critically on severity of the disease, specifically, the status of remaining muscle tissues including respiratory and cardiac muscles. Early treatment provides better protection of patients from disease progression, and less clinical benefit can be achieved with later intervention. The results suggest that design of clinical trial could be critical for exon skipping to achieve high therapeutic value.

Materials and methods

Animals, oligonucleotides and in vivo delivery methods

Ten utrophin-dystrophin-deficient mice (dko) from different age group of 20–29, 30–39, 40–49, 50+ days were used for biweekly 15 mg kg−1 PPMOE23 treatment till mice end point (receiving maximum 11, 10, 9 and 2 injections, respectively). Experiments were approved by Institutional Animal Care and Use Committee Carolinas Medical Center. The PMO (+07–18; 5′-IndexTermGGCCAAACCTCG GCTTACCTGAAAT-3′) conjugated to the peptide (RXRRBR)2XB (R=arginine, X=6-amino hexanoic acid and B=β-alanine) through a non-cleavage amide linker to form a peptide-PMO conjugate (PPMOE23; AVI BioPharma, Bothell, WA, USA) was used against the boundary sequences of exon and intron 23 of the dystrophin gene. PPMOE23 dissolved in 100 μl saline was injected retroorbitally, and 100 μl saline only as control. Mice were killed at desired time points or humane end points, and muscles were snap-frozen in liquid nitrogen-cooled isopentane and stored at −80 °C.

Phenotypic analysis of dko mice

Mice were observed daily for the disease progress and the weight recorded. The end-point guidelines were established for dko mice because of the extremely severe phenotype. The criteria for termination included severely labored breathing, inability to eat or drink, and/or a dramatic decrease of body weight (more than 20% within a week). The surviving mice with the treatment of PPMOE23 were killed when they reached 6-month old. The life span of each mouse was calculated by the termination date. Weights were collected daily and onsets of joint contractures displayed as a pronounced waddling gait and visible kyphosis were recorded. The severity of kyphosis was also measured by radiograph images with piXarray 100 digital specimen radiography system in vitro diagnostic medical device (Bioptics, Inc., Tucson AZ, USA) and analyzed with ImageJ software (v1.42q; http://imagej.nih.gov/ij/). Kyphosis index=the length of AB/The length of CD as illustrated below.34 Ten mice were used for each age group.

Antibodies and immunohistochemistry

Sections of 6 μm were cut from at least two-thirds of muscle length of TA, quadriceps, biceps and gastrocnemius at 100 μm intervals and at least 6 levels from all other muscles including the heart, diaphragm, intercostals and abdominal muscles at 100 μm intervals. The intervening muscle sections were collected for western blot and RT-PCR analyses. The serial sections were stained with rabbit polyclonal antibody P7 against dystrophin, rabbit polyclonal anti-nNOS antibody (Millipore, Billerica, MA, USA), monoclonal clone IIH6C4 antibody against functional α-dystroglycan (Millipore), goat polyclonal anti-β1 integrin antibody (R&D systems, Minneapolis, MN, USA). The primary antibodies were individually detected by Alexa Fluor-594 goat-anti-rabbit IgG, Alexa Fluor-594 goat-anti-mouse IgM, Alexa Fluor-594 donkey-anti-goat IgG (Invitrogen, Eugene, OR, USA). Sections were also stained with hematoxylin and eosin and Masson’s trichrome collagen staining for histological assessment.

Protein extraction and western blot

The collected sections were ground into powder and lysed with 200 μl protein extraction buffer as described previously.13 The protein concentration was quantified by Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Proteins were loaded onto a 4–20% Tris-HCl gradient gel. Samples were electrophoresed at 150 V for 4 h and blotted onto nitrocellulose membrane overnight at 50 V. The membrane was then washed and blocked with 5% skimmed milk and probed with monoclonal antibody NCL-DYS1 against dystrophin rod domain (Vector Labs, Burlingame, CA, USA) overnight. Rabbit anti-α-actin antibody (Sigma, St Louis, MO, USA) was used as loading control. The bound primary antibody was detected by horseradish peroxidase-conjugated goat anti-mouse IgG for dystrophin or horseradish peroxidase-conjugated goat anti-rabbit IgG for α-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and ECL Western Blotting Analysis System (Perkin-Elmer, Waltham, MA, USA). The intensity of the bands obtained from the antisense oligomer-treated mice muscles was measured and compared with that from normal muscles of C57 mice (NIH ImageJ 1.42 software).

RNA extraction and RT-PCR

The collected sections were homogenized in TRIzol (Invitrogen) by using an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany). Total RNA was then extracted and 100 ng of RNA template was used for a 50-μl RT-PCR with RT-PCR Master Mix (USB, Cleveland, OH, USA). The primer sequences for the RT-PCR were Ex20Fo 5′-IndexTermAGAATTCTGCCAATTGCTGAG-3′ and Ex26Ro 5′-IndexTermTCTTCAGCTTGTGTCATCC-3′ for amplification of mRNA from exons 20 to 26. A total of 40 cycles were carried out for the RT-PCR. Bands with the expected size for the transcript with exon 23 deleted were extracted and sequenced. The intensity of the bands was measured with the NIH ImageJ 1.42 software and percentage of exon skipping was calculated with the intensity of the two bands representing both exon 23 unskipped and skipped as 100%.

Measurement of serum creatine kinase and other components

Mouse blood was taken immediately after cervical dislocation and centrifuged at 1500 r.p.m. for 3 min. Serum was separated and stored at −80 °C. The level of serum components was determined by Charles Riverside Laboratories.

Statistical analysis

All data are reported as mean±s.e.m. Statistical differences between treatment groups and control groups were evaluated by Student’s t-test.

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Acknowledgements

This work was supported by the Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation and Carolinas Medical Center, Charlotte, NC, Foundation to Eradicate Duchenne, Department of Defense (W81XWH-09-1-0599), and NIH/NICHD (U54 HD 071601-02). We thank AVI Biopharma (now Sarepta Therapeutics) for the supply of PPMOE23 for this study and Dr Mark Grady (Washington University, St Louis) and Dr Joshua Sanes (Harvard University, Cambridge) for providing mdx female mice heterozygous for utrophin (mdx;utr+/−).

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Correspondence to B Wu or Q L Lu.

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