Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (44)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Aartsma-Rus, A.
Right arrow Articles by van Deutekom, J. C.T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aartsma-Rus, A.
Right arrow Articles by van Deutekom, J. C.T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 8 907-914
DOI: 10.1093/hmg/ddg100
© 2003 Oxford University Press

Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients

Annemieke Aartsma-Rus1, Anneke A.M. Janson1, Wendy E. Kaman1, Mattie Bremmer-Bout1, Johan T. den Dunnen1, Frank Baas2, Gert-Jan B. van Ommen1 and Judith C.T. van Deutekom1,*

1Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands and 2Department of Neurology, Academic Medical Center, Amsterdam, The Netherlands

Received January 13, 2003; Accepted February 14, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
The dystrophin deficiency leading to the severely progressing muscle degeneration in Duchenne muscular dystrophy (DMD) patients is caused by frame-shifting mutations in the DMD gene. We are developing a reading frame correction therapy aimed at the antisense-induced skipping of targeted exons from the pre-mRNA. Despite introducing a (larger) deletion, an in-frame transcript is generated that allows the synthesis of a slightly shorter, but largely functional dystrophin as found in the mostly milder Becker muscular dystrophy (BMD). We have recently demonstrated both the efficacy and high efficiency of the antisense-induced skipping of numerous exons from the DMD transcript in control muscle cells. In principle, this would restore the reading frame in over 75% of the patients reported in the Leiden DMD mutation database. In this study, we in fact demonstrate the broad therapeutic applicability of this strategy in cultured muscle cells from six DMD patients carrying different deletions and a nonsense mutation. In each case, the specific skipping of the targeted exon was induced, restoring dystrophin synthesis in over 75% of cells. The protein was detectable as soon as 16 h post-transfection, then increased to significant levels at the membrane within 2 days, and was maintained for at least a week. Finally, its proper function was further suggested by the restored membranal expression of four associated proteins from the dystrophin–glycoprotein complex. These results document important progress towards a clinically applicable, small-molecule based therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are both caused by mutations in the DMD gene, which is located on the X chromosome and codes for dystrophin (1–6). The severe DMD has an incidence of 1:3500 newborn males. Patients suffer from progressive muscle weakness, are wheelchair-bound before the age of 12 and often die before the third decade of their life (7). The generally milder BMD has an incidence of 1:20 000. BMD patients often remain ambulant for over 40 years and have longer life expectancies when compared with DMD patients (8).

Dystrophin is an essential component of the dystrophin–glycoprotein complex (DGC) which maintains the membrane stability of muscle fibers (9,10). Frame-shifting mutations in the DMD gene result in dystrophin deficiency in muscle cells. This is accompanied by reduced levels of other DGC proteins and results in the severe phenotype found in DMD patients (11,12). Mutations in the DMD gene that keep the reading frame intact, generate shorter but partly functional dystrophins associated with the less severe BMD (13,14).

Despite extensive efforts, no clinically applicable and effective therapy for DMD patients has yet been developed (15), although a delay of the onset of disease manifestations can be achieved by glucocorticoid therapy (16). Promising results have recently been reported by us and others on a genetic therapy aimed at restoring the reading frame of the dystrophin pre-mRNA in cells from the mdx mouse model and DMD patients (17–23). By the targeted skipping of a specific exon, a DMD phenotype can be converted into a milder BMD phenotype. The skipping of an exon can be induced by the binding of antisense oligoribonucleotides (AONs) targeting either one or both of the splice sites, or exon-internal sequences. Since an exon will only be included in the mRNA when both the splice sites are recognized by the spliceosome complex, splice sites are obvious targets for AONs. This was shown to be successful, albeit with variable efficacy and efficiency (17,18,20). We hypothesized that targeting exon-internal sequences might increase specificity and reduce interference with the splicing machinery itself. Some exons have weak splice sites and appear to require binding of a SR protein to an exon recognition sequence (ERS) or an exonic splicing enhancer (ESE) to be properly recognized by the splicing machinery (24). Disruptive point mutations or AONs that block these sequences have been found to result in exon skipping (19,22,24–27,28). Using exon-internal AONs specific for an ERS-like sequence in exon 46, we were previously able to modulate the splicing pattern in cultured myotubes from two different DMD patients with an exon 45 deletion (19). Following AON treatment, exon 46 was skipped, which resulted in a restored reading frame and the induction of dystrophin synthesis in at least 75% of the cells. We have recently shown that exon skipping can also be efficiently induced in human control muscle cells for 15 different DMD exons using exon-internal AONs (23, unpublished results). Of the 15 skippable exons identified in our previous studies, most did not have weak splice sites nor did they contain ERS-like sequences (23). Therefore, we postulate that binding of the AONs to the targeted exon per se may be sufficient to cause exon skipping, either by interfering with one or more components of the splicing machinery or by altering the secondary structure of the RNA in such a manner that the splicing machinery no longer recognizes the exon.

Theoretically, reading frame correction can be achieved by skipping one or two exons flanking a deletion, by skipping in-frame exons containing a nonsense mutation, or by skipping duplicated exons. This would result in proteins similar to those found in various BMD patients (2,29). A survey of the Leiden DMD mutation database shows that we could thus correct over 75% of DMD-causing mutations (30).

Here, we report the actual therapeutic effect of exon skipping for six different mutations. In all patient muscle cell cultures, we were able to restore dystrophin synthesis in 75–80% of treated cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
This study includes six DMD patients affected by different mutations (Table 1). Patient DL 515.2 carries an exon 45–50 deletion; hence exon 51 skipping would be frame-correcting. Patient DL 363.2 has a deletion of exon 45–54; the reading frame for this patient would be corrected by an exon 44 skip. For patient 50685.1, who is affected by an exon 48–50 deletion, reading frame correction requires an exon 51 skip. Patient DL 589.2 has an exon 51–55 deletion; the reading frame would be corrected by an exon 50 skip. Patient 53914.1 carries a single exon 52 deletion. Notably, in this case both the skipping of exon 51 or exon 53 would be frame-correcting. Finally, patient 50423.1 has a deletion of a single basepair in exon 49, at position 7389 on cDNA level, resulting in a frame-shift and a premature stop codon in exon 49. Since exon 49 is an in-frame exon, skipping of this exon would correct the reading frame for this patient.


View this table:
[in this window]
[in a new window]
  		Table 1. Overview of the patients, the AONs and the primer sets used in this study
 
We have previously identified AONs with which the skipping of the mentioned target exons 44, 49, 50, 51 and 53 can be induced at concentrations of 1 µM (23). In subsequent dose–response experiments, however, we have obtained substantial skipping efficiencies with lower concentrations of 500 or 200 nM, and even 100 nM for most AONs (data not shown). This had the extra advantageous effect of lower doses of PEI required for transfection, which significantly reduced the levels of cytotoxicity as found in our earlier transfection experiments. Myotube cultures from the six DMD patients were transfected with the relevant AONs. On average 70–90% of cells showed specific nuclear uptake of fluorescent AONs. RNA was isolated 24 h post-transfection and analysed by RT–PCR (Fig. 1). In all patients, the targeted exons were skipped at high efficiencies, and precisely at the exon boundaries, as confirmed by sequence analysis of the novel shorter transcripts (Fig. 1). For patient 50685.1, an additional transcript fragment was found (Fig. 1C). Sequence analysis showed that this was generated by the activation of a cryptic splice site in exon 51. This was previously also observed in human control cells treated with the same AON (23). Remarkably, low levels of spontaneous exon skipping were observed in untreated cells derived from patients DL 363.2 (exon 44 skip), DL 589.2 (exon 50 skip) and 53914.1 (exon 53 skip). RT–PCR analysis on several larger areas of the DMD gene transcript did not reveal additional, unexpected, aberrant splicing patterns induced by the AON-treatment.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 1. RT–PCR and sequence analysis of dystrophin mRNA fragments of the AON-treated DMD patient myotube cultures, focussing on the regions encompassing the exons targeted for skipping. Shorter, novel transcripts were observed when compared to the untransfected myotube cultures (NT). Sequence analysis confirmed the precise skipping of the targeted exons. An alternatively spliced product, detected for patient 50685.1 (C) was sequenced and found to be derived from activation of a cryptic splice site in exon 51. Shorter fragments, detected in untransfected myotube cultures from DL 363.2 (B), DL 589.2 (D) and 53914.1 (E), were sequenced and found to be the result of the spontaneous skipping of exons 44, 50 and 53, respectively. Note that in some analyses additional fragments, slightly shorter than the wild-type products, were present. This was due to heteroduplex formation; 100 bp, size marker; -RT–PCR, negative control.

 
The resulting in-frame transcripts should restore dystrophin synthesis. Indeed, immuno-histochemical analysis of transfected myotube cultures detected dystrophin in the majority of myotubes for each patient (Fig. 2). The therapeutic efficiency was determined by double-staining, using antibodies against myosin, to identify sufficiently differentiated myotubes, and dystrophin. On average, 75–80% of myosin-positive myotubes showed dystrophin expression. We observed clear membrane-bound dystrophin for patients DL 363.2, DL 589.2 and 53914.1 2 days post-transfection (Fig. 2B, D and E). The presence of dystrophin was confirmed for each patient by western blot analysis (Fig. 3). For patients 50685.1 and DL 363.2 we performed time course experiments, which indicated that dystrophin can be detected as soon as 16 h post-transfection (Fig. 3D) and at increasing levels up to 7 days post-transfection (Fig. 3B). The dystrophin proteins from patients DL 515.2, DL 363.2 and DL 589.2 are significantly shorter than the human control, which is due to the size of the deletion.



View larger version (85K):
[in this window]
[in a new window]
  		Figure 2. Immuno-histochemical analysis of the AON-treated myotube cultures from the six different DMD patients. Cells were stained for myosin to identify fully differentiated myotubes (not shown). Monoclonal antibodies MANDYS1 (middle panel) and Dys2 (right panel) were used to detect dystrophin 1–4 days post-transfection. No dystrophin signals could be detected in untreated cells stained with MANDYS1 (left panel) nor Dys2 (not shown), whereas clear, mainly cytoplasmatic, dystrophin signals could be detected for each patient upon the induced exon skipping. In patients DL 363.2 (B) , DL 589.2 (D) and 53914.1 (E) dystrophin membrane signals could be observed. We note that membrane signals were more often found for Dys2, which recognizes the full-length dystrophin. MANDYS1 recognizes an internal part of dystrophin and is more prone to generate cytoplasmatic signals, since it also detects dystrophin in the first stages of synthesis. Magnification 63x.

 


View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. Western blot analysis of the AON-treated myotube cultures. Monoclonal antibody DY4 was used to detect dystrophin. Protein extracts isolated from human control myotube cultures (HC) were used as a positive control (C and F). To avoid overexposure, this sample was 1–10 diluted. To demonstrate equal loading of protein samples, blots were additionally stained with an antibody against myosin. No, or, as a result of spontaneous exon skipping, very low (B and C) levels of dystrophin were detected in non-transfected myotube cultures (NT). Clear dystrophin signals were observed in AON-treated myotube cultures for each of the patients. For 50685.1 and DL 363.2, a time-course experiment was performed. Dystrophin could be detected 16 h post-transfection and was found at increasing levels at 24 and 48 h post-transfection for 50685.1 (D). For DL 363.2 dystrophin could be detected in increasing levels up to 7 days post-transfection (B). For patients DL 515.2 (A), DL 363.2 (B) and DL 589.2 (E) the detected dystrophin was significantly shorter than the control dystrophin. This is due to the size of the deletions in these patients.

 
For one patient, DL 363.2, we also assessed whether the induction of the dystrophin synthesis resulted in the restoration of the DGC (Fig. 4). Prior to AON treatment we found reduced, mainly cytoplasmatic {alpha}-, ß- and {gamma}-sarcoglycan and ß-dystroglycan signals (30, 30, 40 and 80%, respectively; Fig. 4A). Following AON transfection, increased levels of mainly membrane-bound {alpha}-, ß- and {gamma}-sarcoglycans and ß-dystroglycan were detected in 70, 90, 90 and 80% of the treated myotube cultures, respectively (Fig. 4B).



View larger version (68K):
[in this window]
[in a new window]
  		Figure 4. Immuno-histochemical analysis of four DGC proteins from treated myotube cultures from patient DL 363.2. Cells were stained for myosin to identify sufficiently differentiated myotubes (not shown). Monoclonal antibodies NCL-a-SARC, NCL-b-SARC, NCL-g-SARC and NCL-b-DG were used to detect {alpha}-sarcoglycan, ß-sarcoglycan, {gamma}-sarcoglycan and ß-dystroglycan, respectively. These proteins were detected in reduced percentages (~40%) in untreated myotubes, and were mainly located in the cytoplasm (A). Following AON treatment, however, {alpha}-sarcoglycan was detected in 70%, ß-sarcoglycan was detected in 90%, {gamma}-sarcoglycan was detected in 90% and ß-dystroglycan was detected in 80% of the myotubes, and the proteins were mostly membrane-bound (B). Magnification 63x.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIAL AND METHODS
 REFERENCES
 
Our reading frame correction strategy for DMD patients is aimed at antisense-induced, targeted exon skipping. This would convert a severe DMD phenotype into a mostly milder BMD phenotype. Others and ourselves have already demonstrated that it is feasible to skip various DMD exons using AONs against either splice sites or exon-internal sequences. This was shown in muscle cells derived from mdx mice (18,20,31) and DMD patients carrying a deletion of exon 45, exon 48–50, or within exon 19 (17,19,22), leading to in-frame transcripts and restored dystrophin synthesis. Here, we further determined the broad applicability in six patients, carrying five different deletions and a point mutation in an exon 49 (Table 1). Following AON treatment, we show for each patient the precise skipping of the targeted exon on RNA level, and a dystrophin protein in 75–80% of the treated myotubes. In particular, we here report, for the first time, the application of a single AON treatment (i.e. the induced skipping of exon 51) to correct the reading frame for several different deletions.

Interestingly, the levels of exon skipping observed in the DMD patient cells are significantly higher than those previously obtained in human control cells (23). Typically, the novel skip transcript is the major product. This can be explained by the action of the nonsense-mediated decay (NMD) process (25,32). In control cells, the skip of an out-of-frame exon results in an out-of-frame transcript, which will be susceptible to NMD. In patient cells, the skip of a target exon results in an in-frame transcript that would be resistant to NMD and thus more stable than the out-of-frame transcript originally present.

For three of the patients (DL 363.2, DL 589.2 and 53914.1) we detected low levels of spontaneous skipping of exons 44, 50 and 53 in untreated cells. This phenomenon has previously also been described for so-called revertant muscle fibres (33–35). These dystrophin positive fibres are present in low amounts (2–10%) in DMD muscles, and are considered to be the result of secondary somatic mutations and/or alternative splicing that restore the reading frame. The existence of revertant fibres has been suggested to correlate with the severity of the disease (36,37).

Restoration of the dystrophin synthesis could be detected as soon as 16 h post-transfection. At 2 days post-transfection, dystrophin was detected at the membrane, indicating that these novel BMD-like proteins are likely to be in part functional. Furthermore, we show that restoration of the dystrophin synthesis appears to re-establish the formation of the dystrophin-glycoprotein complex.

In patients DL 363.2 and DL 589.2, the targeted exon skipping enlarged the deletions to span exons 44–54 and 50–55, respectively. So far, these deletions, have not been reported in DMD or BMD patients. This means that they either do not exist or generate a very mild phenotype not diagnosed as BMD. Considering both the large variety of BMD mutations and the markedly lower incidence of BMD observed, we consider the last explanation more plausible than the first. The out-of-frame deletions from patients DL 515.1, 50685.1 and 50423.1 were converted into in-frame deletions as observed in BMD patients carrying deletions of exon 45–51, exon 48–51 and exon 49 (30,38–40). It is noteworthy that the exon 48–51 deletion has even been described in an asymptomatic person (40). On the other hand, however, there are also DMD patients carrying such deletions (38,41–43). Since most of these theoretically in-frame deletions have been detected on DNA level only, we hypothesize that the dystrophin deficiency in these DMD patients may be caused by additional aberrant splicing patterns on RNA level, resulting in an out-of-frame transcript.

With the advances we report here, proof of principle is provided, in human patient cells, for the feasibility to correct over 75% of the mutations reported in the Leiden DMD mutation database (30). Our results indicate that, provided that a suitable means of administration for the AONs is developed, antisense-induced reading frame correction will be a promising therapeutic approach for many DMD patients carrying different deletions and point mutations. Towards the establishment of clinical trials, we are currently investigating and optimizing delivery methods in muscle tissue of mice in vivo.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
REFERENCES
 
AONs and primers
The AONs applied (Table 1) were previously described (23). They contain a 5' fluorescein group (6-FAM), a full-length phosphorothioate backbone and 2'-O-methyl modified ribose molecules (Eurogentec, Belgium). To avoid interference with the fluorescent signals of the secondary antibodies, unlabelled AONs were used for immuno-histochemical analyses. Primers for RT–PCR analysis (sequences available upon request) were synthesized by Eurogentec (Belgium) or by Isogen Bioscience BV (The Netherlands).

Myogenic cell cultures and AON transfections
Primary human myoblasts from patients DL 515.2 (deletion exon 45–50), DL 363.2 (deletion exon 45–54), 50685.1 (deletion exon 48–50), DL 589.2 (deletion exon 51–55) and 53914.1 (deletion exon 52) were isolated from a muscle biopsy and cultured as described (44). Cultures were seeded in collagen pre-coated flasks and plates (Vitrogen 100, Cohesion). Myotubes were obtained from confluent myoblast cultures, following 7–14 days of serum deprivation. They were subsequently transfected using polyethylenimine (PEI) for 3 h in low-serum medium, according to the manufacturer's instructions (ExGen500; MBI Fermentas), and with 3.5 µl PEI applied per µg of transfected AON. For RT–PCR analysis, concentrations of 500 nM AON were used. At this concentration highest skipping levels can be obtained, albeit with moderate levels of cell death. Since for immunohistochemical and western blot analysis more viable myotubes are required, concentrations of 200 nM were applied.

For patient 50423.1, who carries a point mutation in exon 49, only fibroblasts were available. Following infection (MOI 50–100) with an adenoviral vector containing the MyoD gene (Ad50MyoD), the fibroblasts were forced into myogenesis according to protocols described previously (45–47). Two hours post-infection the medium was replaced by low serum medium, and cells were incubated for 8–10 days until myotubes were formed. Transfection conditions were identical to those described above.

RNA isolation and RT–PCR analysis
At 24 h post-transfection, total RNA was isolated from the myotube cultures (RNA-Bee RNA isolation solvent, Campro Scientific, The Netherlands). Aliquots of 300 ng of total RNA were used for RT–PCR analysis using C. therm polymerase (Roche Diagnostics, The Netherlands) in a 20 µl reaction at 60°C for 30 min, primed with different DMD gene-specific reverse primers (Table 1). Primary PCRs were performed by 20 cycles of 94°C (40 s), 60°C (40 s) and 72°C (60 s). One microlitre of these reactions was then reamplified in nested PCRs by 32 cycles of 94°C (40 s), 60°C (40 s) and 72°C (60 s). PCR products were analysed on 1.5 or 2% agarose gels. It is noteworthy that no evidence for a significant preference for the amplification of shorter fragments was obtained in PCR analyses on a defined series of mixtures of known quantities of the normal and shorter transcript fragments (data not shown).

Sequence analysis
RT–PCR products were isolated from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Direct DNA sequencing was carried out by the Leiden Genome Technology Center (LGTC) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems) and analysed on an ABI 3700 Sequencer (PE Applied Biosystems).

Protein isolation and western blot analysis
Protein extracts were isolated from treated myotube cultures (25 cm2 flasks), using 150 µl of treatment buffer (75 mM Tris–HCl pH 6.8, 15% SDS, 5% ß-mercaptoethanol, 2% glycerol, 0.001% bromophenol blue), at 2–4 days post-transfection depending on the survival rate of the myotubes. For the time course experiments, protein extracts were isolated 4, 8, 16, 24 and 48 h post-transfection (for patient 50685.1) or at 2, 4 and 7 days post-transfection (for patient DL 363.2). Polyacrylamide gel electrophoresis and western blotting were performed as described by Anderson et al., with some minor adjustments (48). Briefly, samples (75 µl) were run overnight at 4°C on a 4–7% polyacrylamide gradient gel. Gels were blotted to nitrocellulose for 5–6 h at 4°C. Blots were blocked for 1 h with 5% non-fat dried milk in TBST buffer (10 mM Tris–HCl, 0.15 M NaCl, 0.5% Tween 20, pH 8) followed by an overnight incubation with NCL-DYS2 (which recognizes dystrophin) diluted 1:50. HRP-conjugated anti-mouse (Santa Cruz) diluted 1:10 000 was used as a secondary antibody. Immuno-reactive bands were visualized using Lumi-Lightplus Western Blotting Substrate (Roche Diagnostics, The Netherlands) and scanned with a Lumi-Imager (Roche Diagnostics, The Netherlands).

Immuno-histochemical analysis
Treated myotube cultures were fixed in -20°C methanol at 1–4 days post-transfection, depending of the survival rate of the myotubes. Prior to reaction with the different antibodies, the cells were incubated for 1 h in a blocking solution containing 5% horse serum (Gibco BRL) and 0.05% Tween-20 (Sigma) in PBS (Gibco BRL). All antibodies used were diluted in this blocking solution. The following antibodies were applied: desmin polyclonal antibody (ICN Biomedicals) diluted 1:100, myosin monoclonal antibody diluted 1:100 (MF20; Developmental Studies Hybridoma Bank, University of Iowa), myosin polyclonal antibody L53 diluted 1:100 (a gift from Dr M. van den Hoff, AMC, The Netherlands), MANDYS1 (a gift from Dr G. Morris, North East Wales Institute, UK) diluted 1:10 and NCL-DYS2 (Novacastra Laboratories Ltd) diluted 1:10 to detect dystrophin, NCL-a-SARC (Novacastra Laboratories Ltd) diluted 1:75, NCL-b-SARC (Novacastra Laboratories Ltd) diluted 1:50, NCL-g-SARC (Novacastra Laboratories Ltd) diluted 1:50 and NCL-b-DG (Novacastra Laboratories Ltd) diluted 1:50 to detect {alpha}-sarcoglycan, ß-sarcoglycan, {gamma}-sarcoglycan and ß-dystroglycan, respectively. After 1 h incubation, slides were rinsed and incubated for 1 h with the secondary antibodies Alexa Fluor 594 goat anti-rabbit conjugate diluted 1:1000 or Alexa Fluor 488 goat anti-mouse conjugate diluted 1:250 (Molecular Probes Inc). The slides were analysed using a Leica confocal microscope equipped with epifluorescence optics. Digital images were captured using a CCD camera (Photometrics).


   ACKNOWLEDGEMENTS
 
We would like to thank Ellen Sterrenburg for performing part of the MyoD infections, and Ruud Wolterman for assistance with patient cell cultures. This work was financially supported by the Duchenne Parent Project (The Netherlands), the Princes Beatrix Fund (The Netherlands) and the EU (QLG2-CT-1999-00920).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +31 715276080; Fax: +31 715276075; Email: deutekom{at}lumc.nl Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 

  1. Hoffman, E.P., Brown, R.H. Jr and Kunkel, L.M. (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51, 919–928.[CrossRef][ISI][Medline]

  2. Hoffman, E.P., Fischbeck, K.H., Brown, R.H., Johnson, M., Medori, R., Loike, J.D., Harris, J.B., Waterston, R., Brooke, M., Specht, L. et al. (1988) Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. New Engl. J. Med., 318, 1363–1368.[Abstract]

  3. Den Dunnen, J.T., Grootscholten, P.M., Bakker, E., Blonden, L.A., Ginjaar, H.B., Wapenaar, M.C., van Paassen, H.M., van Broeckhoven, C., Pearson, P.L. and van Ommen, G.J. (1989) Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet., 45, 835–847.[ISI][Medline]

  4. Koenig, M., Beggs, A.H., Moyer, M., Scherpf, S., Heindrich, K., Bettecken, T., Meng, G., Muller, C.R., Lindlof, M., Kaariainen, H. et al. (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am. J. Hum. Genet., 45, 498–506.[ISI][Medline]

  5. Tuffery-Giraud, S., Chambert, S., Demaille, J. and Claustres, M. (1999) Point mutations in the dystrophin gene: evidence for frequent use of cryptic splice sites as a result of splicing defects. Hum. Mutat., 14, 359–368.[CrossRef][ISI][Medline]

  6. Prior, T.W., Bartolo, C., Pearl, D.K., Papp, A.C., Snyder, P.J., Sedra, M.S., Burghes, A.H. and Mendell, J.R. (1995) Spectrum of small mutations in the dystrophin coding region. Am. J. Hum. Genet., 57, 22–33.[ISI][Medline]

  7. Moser, H. (1984) Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention. Hum. Genet., 66, 17–40.[CrossRef][ISI][Medline]

  8. Emery, A.E. (2002) The muscular dystrophies. Lancet, 359, 687–695.[CrossRef][ISI][Medline]

  9. Yoshida, M. and Ozawa, E. (1990) Glycoprotein complex anchoring dystrophin to sarcolemma. J. Biochem. (Tokyo), 108, 748–752.[Abstract/Free Full Text]

  10. Ervasti, J.M. and Campbell, K.P. (1991) Membrane organization of the dystrophin–glycoprotein complex. Cell, 66, 1121–1131.[CrossRef][ISI][Medline]

  11. Di Blasi, C., Morandi, L., Barresi, R., Blasevich, F., Cornelio, F. and Mora, M. (1996) Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers. Acta Neuropathol. (Berl), 92, 369–377.[CrossRef][Medline]

  12. Ervasti, J.M., Ohlendieck, K., Kahl, S.D., Gaver, M.G. and Campbell, K.P. (1990) Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature, 345, 315–319.[CrossRef][Medline]

  13. Matsumura, K., Burghes, A.H., Mora, M., Tome, F.M., Morandi, L., Cornello, F., Leturcq, F., Jeanpierre, M., Kaplan, J.C., Reinert, P. et al. (1994) Immunohistochemical analysis of dystrophin-associated proteins in Becker/Duchenne muscular dystrophy with huge in-frame deletions in the NH2-terminal and rod domains of dystrophin. J. Clin. Invest., 93, 99–105.[ISI][Medline]

  14. Monaco, A.P., Bertelson, C.J., Liechti-Gallati, S., Moser, H. and Kunkel, L.M. (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics, 2, 90–95.[CrossRef][Medline]

  15. Clemens, P.R. and Duncan, F.J. (2001) Progress in gene therapy for Duchenne muscular dystrophy. Curr. Neurol. Neurosci. Rep., 1, 89–96.[Medline]

  16. Khan, M.A. (1993) Corticosteroid therapy in Duchenne muscular dystrophy. J. Neurol. Sci., 120, 8–14.[CrossRef][ISI][Medline]

  17. De Angelis, F.G., Sthandier, O., Berarducci, B., Toso, S., Galluzzi, G., Ricci, E., Cossu, G. and Bozzoni, I. (2002) Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48–50 DMD cells. Proc. Natl Acad. Sci. USA, 99, 9456–9461.[Abstract/Free Full Text]

  18. Mann, C.J., Honeyman, K., Cheng, A.J., Ly, T., Lloyd, F., Fletcher, S., Morgan, J.E., Partridge, T.A. and Wilton, S.D. (2001) Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc. Natl Acad. Sci. USA, 98, 42–47.[Abstract/Free Full Text]

  19. van Deutekom, J.C., Bremmer-Bout, M., Janson, A.A., Ginjaar, I.B., Baas, F., den Dunnen, J.T. and van Ommen, G.J. (2001) Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum. Mol. Genet., 10, 1547–1554.[Abstract/Free Full Text]

  20. Wilton, S.D., Lloyd, F., Carville, K., Fletcher, S., Honeyman, K., Agrawal, S. and Kole, R. (1999) Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides. Neuromusc. Disord., 9, 330–338.[CrossRef][ISI][Medline]

  21. Dunckley, M.G., Manoharan, M., Villiet, P., Eperon, I.C. and Dickson, G. (1998) Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum. Mol. Genet., 7, 1083–1090.[Abstract/Free Full Text]

  22. Takeshima, Y., Wada, H., Yagi, M., Ishikawa, Y., Minami, R., Nakamura, H. and Matsuo, M. (2001) Oligonucleotides against a splicing enhancer sequence led to dystrophin production in muscle cells from a Duchenne muscular dystrophy patient. Brain Dev., 23, 788–790.[CrossRef][ISI][Medline]

  23. Aartsma-Rus, A., Bremmer-Bout, M., Janson, A., den Dunnen, J., van Ommen, G. and van Deutekom, J. (2002) Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromusc. Disord., 12, S71.

  24. Shiga, N., Takeshima, Y., Sakamoto, H., Inoue, K., Yokota, Y., Yokoyama, M. and Matsuo, M. (1997) Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy. J. Clin. Invest., 100, 2204–2210.[ISI][Medline]

  25. Cartegni, L., Chew, S.L. and Krainer, A.R. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet., 3, 285–298.[CrossRef][ISI][Medline]

  26. Schaal, T.D. and Maniatis, T. (1999) Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA. Mol. Cell. Biol., 19, 261–273.[Abstract/Free Full Text]

  27. Takeshima, Y., Nishio, H., Sakamoto, H., Nakamura, H. and Matsuo, M. (1995) Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. J. Clin. Invest., 95, 515–520.[ISI][Medline]

  28. Pramono, Z.A., Takeshima, Y., Alimsardjono, H., Ishii, A., Takeda, S. and Matsuo, M. (1996) Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence. Biochem. Biophys. Res. Commun., 226, 445–449.[CrossRef][ISI][Medline]

  29. Koenig, M., Monaco, A.P. and Kunkel, L.M. (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell, 53, 219–226.[CrossRef][ISI][Medline]

  30. Den Dunnen, J. (1996) The Leiden Muscular Dystrophy pages; www.dmd.nl.

  31. Mann, C.J., Honeyman, K., McClorey, G., Fletcher, S. and Wilton, S.D. (2002) Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. J. Gene Med., 4, 644–654.[CrossRef][ISI][Medline]

  32. Kerr, T.P., Sewry, C.A., Robb, S.A. and Roberts, R.G. (2001) Long mutant dystrophins and variable phenotypes: evasion of nonsense-mediated decay? Hum. Genet., 109, 402–407.[CrossRef][ISI][Medline]

  33. Klein, C.J., Coovert, D.D., Bulman, D.E., Ray, P.N., Mendell, J.R. and Burghes, A.H. (1992) Somatic reversion/suppression in Duchenne muscular dystrophy (DMD): evidence supporting a frame-restoring mechanism in rare dystrophin-positive fibers. Am. J. Hum. Genet., 50, 950–959.[ISI][Medline]

  34. Sherratt, T.G., Vulliamy, T., Dubowitz, V., Sewry, C.A. and Strong, P.N. (1993) Exon skipping and translation in patients with frameshift deletions in the dystrophin gene. Am. J. Hum. Genet., 53, 1007–1015.[Medline]

  35. Lu, Q.L., Morris, G.E., Wilton, S.D., Ly, T., Artem'yeva, O.V., Strong, P. and Partridge, T.A. (2000) Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion. J. Cell Biol., 148, 985–996.[Abstract/Free Full Text]

  36. Nicholson, L.V., Johnson, M.A., Bushby, K.M. and Gardner-Medwin, D. (1993) Functional significance of dystrophin positive fibres in Duchenne muscular dystrophy. Arch. Dis. Child, 68, 632–636.[Abstract]

  37. Vainzof, M., Passos-Bueno, M.R., Takata, R.I., Pavanello Rde, C. and Zatz, M. (1993) Intrafamilial variability in dystrophin abundance correlated with difference in the severity of the phenotype. J. Neurol. Sci., 119, 38–42.[CrossRef][ISI][Medline]

  38. Singh, V., Sinha, S., Mishra, S., Chaturvedi, L.S., Pradhan, S., Mittal, R.D. and Mittal, B. (1997) Proportion and pattern of dystrophin gene deletions in north Indian Duchenne and Becker muscular dystrophy patients. Hum. Genet., 99, 206–208.[CrossRef][ISI][Medline]

  39. Melacini, P., Fanin, M., Danieli, G.A., Fasoli, G., Villanova, C., Angelini, C., Vitiello, L., Miorelli, M., Buja, G.F., Mostacciuolo, M.L. et al. (1993) Cardiac involvement in Becker muscular dystrophy. J. Am. Coll. Cardiol., 22, 1927–1934.[Abstract]

  40. Melis, M.A., Cau, M., Muntoni, F., Mateddu, A., Galanello, R., Boccone, L., Deidda, F., Loi, D. and Cao, A. (1998) Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families. Eur. J. Paediatr. Neurol., 2, 255–261.[CrossRef][Medline]

  41. Onengut, S., Kavaslar, G.N., Battaloglu, E., Serdaroglu, P., Deymeer, F., Ozdemir, C., Calafell, F. and Tolun, A. (2000) Deletion pattern in the dystrophin gene in Turks and a comparison with Europeans and Indians. Ann. Hum. Genet., 64, 33–40.[CrossRef][ISI][Medline]

  42. Rosenberg, C., Navajas, L., Vagenas, D.F., Bakker, E., Vainzof, M., Passos-Bueno, M.R., Takata, R.I., Van Ommen, G.J., Zatz, M. and Den Dunnen, J.T. (1998) Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization. Neuromusc. Disord., 8, 447–452.[CrossRef][ISI][Medline]

  43. Sertic, J., Barisic, N., Sostarko, M., Brzovic, Z. and Stavljenic-Rukavina, A. (1997) Deletion screening of the Duchenne/Becker muscular dystrophy gene in Croatian population. Coll. Antropol., 21, 151–156.[ISI][Medline]

  44. Rando, T.A. and Blau, H.M. (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol., 125, 1275–1287.[Abstract/Free Full Text]

  45. Murry, C.E., Kay, M.A., Bartosek, T., Hauschka, S.D. and Schwartz, S.M. (1996) Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. J. Clin. Invest., 98, 2209–2217.[ISI][Medline]

  46. Roest, P.A., van der Tuijn, A.C., Ginjaar, H.B., Hoeben, R.C., Hoger-Vorst, F.B., Bakker, E., den Dunnen, J.T. and van Ommen, G.J. (1996) Application of in vitro Myo-differentiation of non-muscle cells to enhance gene expression and facilitate analysis of muscle proteins. Neuromusc. Disord., 6, 195–202.[CrossRef][ISI][Medline]

  47. Havenga, M.J., Lemckert, A.A., Ophorst, O.J., van Meijer, M., Germeraad, W.T., Grimbergen, J., van Den Doel, M.A., Vogels, R., van Deutekom, J., Janson, A.A. et al. (2002) Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. J. Virol., 76, 4612–4620.[Abstract/Free Full Text]

  48. Anderson, L.V. and Davison, K. (1999) Multiplex Western blotting system for the analysis of muscular dystrophy proteins. Am. J. Pathol., 154, 1017–1022.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
P. A. C. 't Hoen, E. J. de Meijer, J. M. Boer, R. H. A. M. Vossen, R. Turk, R. G. H. J. Maatman, K. E. Davies, G.-J. B. van Ommen, J. C. T. van Deutekom, and J. T. den Dunnen
Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene
J. Biol. Chem., February 29, 2008; 283(9): 5899 - 5907.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
J. C. van Deutekom, A. A. Janson, I. B. Ginjaar, W. S. Frankhuizen, A. Aartsma-Rus, M. Bremmer-Bout, J. T. den Dunnen, K. Koop, A. J. van der Kooi, N. M. Goemans, et al.
Local Dystrophin Restoration with Antisense Oligonucleotide PRO051
N. Engl. J. Med., December 27, 2007; 357(26): 2677 - 2686.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
A. Aartsma-Rus and G.-J. B. van Ommen
Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications
RNA, October 1, 2007; 13(10): 1609 - 1624.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
A AARTSMA-RUS, A.A.M JANSON, J.A HEEMSKERK, C.L DE WINTER, G.-J.B VAN OMMEN, and J.C.T VAN DEUTEKOM
Therapeutic Modulation of DMD Splicing by Blocking Exonic Splicing Enhancer Sites with Antisense Oligonucleotides
Ann. N.Y. Acad. Sci., October 1, 2006; 1082(1): 74 - 76.
[Abstract] [Full Text] [PDF]

Home page
 Postgrad. Med. J.Home page
S M Selkirk
Gene therapy in clinical medicine
Postgrad. Med. J., October 1, 2004; 80(948): 560 - 570.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (44)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Aartsma-Rus, A.
Right arrow Articles by van Deutekom, J. C.T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aartsma-Rus, A.
Right arrow Articles by van Deutekom, J. C.T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?