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DMD is a fatal myodegenerative condition that results from either non-sense or frame-shifting mutations in the dystrophin gene, leading to adearth of dystrophin protein in the muscle fibers1. Dystrophin-negativemuscle fibers are sensitive to a variety of types of stress, and undergorepetitive cycles of damage and repair as a consequence of day-to-dayactivity. The muscles eventually exhaust their regenerative capacity andmuscle fibers are gradually replaced by adipose and connective tissues.Patients usually die from respiratory or cardiac failure before the thirddecade of life. This disease cannot be eliminated by genetic screeningbecause one case in three is caused by a de novo mutation2, so an effectivetherapy will always be needed.

Simple replacement of the protein-coding region of the dystrophingene has proven problematic3–6. An alternate option is suggested by thefact that the majority of mutations in both DMD and the milder Beckermuscular dystrophy (BMD) occur within the spectrin-like rod domain ofthe dystrophin gene, but mutations in BMD do not disrupt the readingframe, thus permitting production of partially functional dystrophinprotein7. This raises the possibility of a general therapy by skipping exonsbearing mutations and exons whose removal restores the open readingframe. Such transcripts can be translated into a shortened dystrophinprotein retaining crucial functional domains. Recent work by our groupand others has shown, in in vitro experiments using myogenic cell cul-ture, that this can be achieved by antisense strategies targeted to themutated exon(s)8–10. Here we show that the mutated exon 23 in the mdxmice can be specifically removed by a particular, previously identified2OMeAO, restoring dystrophin expression to levels comparable to thoseof normal muscle fibers. More importantly, single intramuscular

injections of 2OMeAO resulted in expression of dystrophin for morethan 3 months and improvement of the physiological functions of themuscles. These results show the real feasibility of using this strategy forclinical treatment of DMD, as well as other diseases in which exon splic-ing might be used to obviate nonsense and frame-shift mutations.

RESULTS2OMeAO and F127 induce dystrophin expressionOur previous gene delivery studies identified a nonionic block copolymer,F127, that markedly improves transgene expression by naked plasmidDNA without causing muscle damage11. We used the potent transfectionproperties of F127 to improve the efficiency of delivery of 2OMeAO to thetibialis anterior muscle of 4-week-old mdx mice. Tibialis anterior musclesinjected with 5 µg of 2OMeAO together with 10 µg of F127, in a total vol-ume of 30 µl, rapidly accumulated dystrophin. By immunostaining, wedetected large numbers of fibers expressing normal levels of dystrophin 2weeks after intramuscular injection (Fig. 1a). More than 400 dystrophin-positive fibers per section were found consistently (Fig. 1b). Most of thesewere stained at an intensity comparable with that in costained sections ofwild-type (C57Bl/10ScSn; designated C57) muscle, but some were stainedless strongly. A maximum of 35 positive ‘revertant’ fibers12 were found incontralateral muscles injected with ‘sense’ oligonucleotide or with salineonly. This same formulation of 2OMeAO or control sense construct withF127 was used in all subsequent experiments.

Restoration of dystrophin expression in old miceAge-related diminution of efficiency is a common feature of both viral

1Muscle Cell Biology, MRC Clinical Science Centre, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK. 2Australian Neuromuscular Research Institute,Centre for Neuromuscular and Neurological Disorders, QEII Medical Centre University of Western Australia, Nedlands 6009, Western Australia. 3University ofHertfordshire, Department of Biosciences, College Lane, Hatfield, Herts, AL10 9AB, UK. 4MRIC Biochemistry Group, The North East Wales Institute, Plas Coch, MoldRoad, Wrexham, LL11 2AW, UK. 5Department of Immunology, Division of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK. Correspondence should be addressed to T.A.P. (terence.partridge@csc.mrc.ac.uk) or Q.L.L. (qi.long-lu@csc.mrc.ac.uk).

Functional amounts of dystrophin produced by skippingthe mutated exon in the mdx dystrophic mouseQi Long Lu1, Christopher J Mann2, Fang Lou3, George Bou-Gharios1, Glenn E Morris4, Shao-an Xue5,Sue Fletcher2, Terence A Partridge1 & Stephen D Wilton2

As a target for gene therapy, Duchenne muscular dystrophy (DMD) presents many obstacles but also an unparalleled prospect forcorrection by alternative splicing. The majority of mutations in the dystrophin gene occur in the region encoding the spectrin-likecentral rod domain, which is largely dispensable. Thus, splicing around mutations can generate a shortened but in-frame transcript,permitting translation of a partially functional dystrophin protein. We have tested this idea in vivo in the mdx dystrophic mouse(carrying a mutation in exon 23 of the dystrophin gene) by combining a potent transfection protocol with a 2-O-methylatedphosphorothioated antisense oligoribonucleotide (2OMeAO) designed to promote skipping of the mutated exon. The treated miceshow persistent production of dystrophin at normal levels in large numbers of muscle fibers and show functional improvement of thetreated muscle. Repeated administration enhances dystrophin expression without eliciting immune responses. Our data establishesthe realistic practicality of an approach that is applicable, in principle, to a majority of cases of severe dystrophinopathy.

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and nonviral gene delivery into skeletal muscle,especially where dystrophyis associated with increased extracellular matrix. However, the number ofdystrophin-positive fibers induced by 2OMeAO was similar in tibialis ante-rior muscles of mice aged 6 months,4 weeks and 2 weeks,with averages of477,504 and 433, respectively and accounting for between 15 and 21% ofthe total muscle fibers of the cross-sectional area (CSA; Fig. 1a,b). In miceof all ages, the majority of dystrophin-positive fibers extended more than

300 µm in length, although only a small proportion were stained for theentire two-thirds of the muscle length examined. However, expression ofdystrophin in fibers around the injection site was more homogeneous in2-week-old mouse muscles than those in older mice (Fig. 1a).

2OMeAO-induced dystrophin retains crucial domainsOur previous studies showed that 2OMeAO induces skipping

predominantly between exons 22 and 24,producing an in-frame transcript detected byRT-PCR with primers amplifying sequencesbetween exons 20 and 26 (ref. 8). This impliesthat only the 71 amino acids encoded byexon 23 are lost. However, a large proportionof the 79 exons in the rod region of dys-trophin are bounded by complete codontriplets. It is therefore possible that smallexons outside the PCR-surveyed region couldbe lost from the protein product. Such alter-ations with limited changes in protein sizewould not be detected by western blottingwith single antibody.

We therefore looked for any significantskipping of exons outside the targeted regionby immunofluorescence epitope mapping onserial sections with a panel of antibodies spe-cific for individual exons or exon groups12.The same set of dystrophin-positive fiberswas recognized by all antibodies, includingthose specific for exons in N-terminal, cys-teine-rich and C-terminal domains (Fig. 2a,b). The occasional apparent lack ofone-to-one correspondence between indi-vidual fibers in this series partly reflects

a b

*

Figure 1 Induction of dystrophin by 20MeAO and F127 in mice of various ages. (a) Dystrophin expression 2 weeks after intramuscular injection of2OMeAO at 2, 4 and 24 weeks of age. Control was injected with sense oligonucleotide at 24 weeks. Dystrophin expression is more homogeneous in fibersaround the injection site (arrowhead) in 2-week-old mouse. (b) Numbers of dystrophin-positive fibers per muscle section 2 weeks after intramuscularinjection of 2OMeAO at the ages of 2, 4 and 24 weeks. Control muscles (Cont) from 24-week-old mdx mice were injected with sense oligonucleotide. *, P < 0.001; n = 4–6 mice; data presented as mean ± s.e.m.

a

b

Figure 2 Detection of dystrophin and dystrophinprotein complexes. Exon mapping of 2OMeAO-induced dystrophin (a,b) and detection ofdystrophin protein complexes (b) in serial sections.Top rows of each panel show sections from controltibialis anterior muscle of C57 mice, with the nameand exon (E) specificity of the antibody at the top.Middle rows show tibialis anterior muscle of mdxmice 4 weeks after injection with 2OMeAO, at theage of 6 weeks. Bottom rows show senseoligonucleotide–treated mdx tibialis anteriormuscle. Images are arranged from N-terminal (left)to C-terminal (right) exons along the dystrophingene (not in order of serial sections).

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longitudinal variation in dystrophin content, as it is also seen withany single antibody. Overall, we conclude that the majority of the2OMeAO-induced dystrophin contains all of the exons for which wehave specific antibodies. This contrasts with the spontaneouslyexpressed ‘revertant dystrophins’, which commonly lose epitopes cor-responding to more than 20 exons12.

2OMeAO-induced dystrophin also retained the functionaldomains responsible for assembly of the dystrophin protein com-plex at the surface of the muscle fiber. Immunofluorescent studieson serial sections showed restoration of dystroglycans and sarcogly-cans at the membranes of fibers expressing truncated dystrophin;these glycans were maintained during the period in which dys-trophin was expressed (up to 3 months; Fig. 2b). Restoration ofmembrane localization of neuronic nitroxidase (nNOS) wasdelayed: staining was seen only weakly at 1 week, but clear mem-brane localization was seen 2 weeks after intramuscular injection(Fig. 3).

Duration of 2OMeAO-induced dystrophin expressionClinical application of antisense therapy to DMD patients wouldrequire persistent expression of the induced dystrophin. In 4-week-old mdx mice, both the number of fibers expressing dystrophin andthe total amount of protein per muscle, as detected by western blots,showed a peak of expression 2–4 weeks after a single intramuscularinjection and persisted at readily detectable levels up to 2 months(Fig. 4a–c). By 3 months, both immunostaining and western blot-ting showed a considerable drop in dystrophin content, although itremained detectable (Fig. 4a–c). The amount of protein expressedin the mdx muscles, measured by signal intensity of the western blot2–4 weeks after 2OMeAO injection, was about 20% of that in nor-mal C57 muscle. Because this is comparable to the percentage ofdystrophin-positive fibers, we inferred that these fibers containclose to normal levels of dystrophin. The size of the induced dys-trophin protein was, aside from the minor exon loss predicted,indistinguishable from that of normal dystrophin and remained soat all time points. Essentially the same temporal pattern of rise andfall in dystrophin expression was observed after injection of2OMeAO, irrespective of age.

Partial restoration of physiological functionTo test for physiological function of 2OMeAO-induced dystrophinand associated protein complexes, we examined the generation ofisometric force in 2OMeAO-treated muscles compared with thesense oligonucleotide–injected contralateral muscles of 6-week-oldmdx and age-matched C57 normal mice. We found no significantdifference in weight or CSA between 2OMeAO-injected and thecontralateral tibialis anterior muscles (Fig. 5a). The normalizedmaximum isometric tetanic force in all muscles injected with

Figure 3 Detection of membrane-localized nNOS in fibers expressing dystrophin. Right panels, tibialis anterior muscle of a 4-week-old mdx mouse, 2 weeksafter 2OMeAO injection. Left panels, tibialis anterior muscle of C57mouse used as positive control. Most dystrophin-positive fibers stained with P6 antibodyin the 2OMeAO-treated mdx muscle show membrane staining for nNOS. Nuclei were counterstained with DAPI (blue).

a

b c

Figure 4 Duration of dystrophin expression in tibialis anterior muscles of 4-week-old mdx mice 1, 2, 4, 8 and 12 weeks after injection of 2OMeAO orsense oligonucleotide (control). (a) Dystrophin expression. (b) Numbers ofdystrophin-positive fibers. *, P < 0.01; n = 4–6 mice; data presented asmean ± s.e.m. (c) Western blot of dystrophin expression in 2OMeAO-injectedmdx tibialis anterior muscles. Control was blotted 4 weeks after injectionwith sense oligo. Positive control is an 8-week-old C57 mouse. Sampleswere taken from two mice per time point, except for positive control.

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2OMeAO was significantly improved (P < 0.01, n = 5) comparedwith the contralateral muscles, although distinctly lower than thatof the muscle from normal C57 mice (Fig. 5b). Injection of2OMeAO, however, produced no clear benefit in terms of fatigueresistance, with tetanic force declining at the same rate as in senseoligonucleotide–injected mdx muscles (data not shown). This might

reflect the presence of dystrophin-negative segments in most fibers,which may leave them vulnerable to this form of work.Immunohistochemical staining of all muscles after force testingconfirmed that the number of fibers expressing dystrophin rangedfrom 389 to 458 fibers (Fig. 5c). These fibers had slightly diffusedmembrane staining, perhaps reflecting their stressed state after 3 hin buffer.

Lack of autoimmune responseThe 2OMeAO-induced dystrophin is close to full length, presum-ably lacking only exon 23. Thus, there is the possibility of autoim-mune responses against epitopes that are not normally expressed.After a single injection of 2OMeAO, tibialis anterior musclesshowed no signs of excessive degeneration or infiltration, above lev-els seen in control mdx muscle, by monocytes or CD4+ or CD8+

lymphocytes. In fact, only the occasional, isolated CD4+ or CD8+

lymphocyte was identified in most areas containing dystrophin-expressing fibers up to 3 months later (Fig. 6a). Readministration of2OMeAO 3 months after the initial intramuscular injection simi-larly showed no augmentation of any category of inflammatory cell.Moreover, the number of muscle fibers expressing dystrophin

a

b c

Figure 5 Physiological analysis of tibialis anterior muscles from C57 and mdx mice treated with 2OMeAO (mdx-T) or sense (mdx-C)oligonucleotides. (a) Quantitative data, presented as mean ± s.e.m.Normalized maximum tetanic force is expressed as percentage of C57control. *, P < 0.01; n = 5. (b) Normalized tetanic force in one mdxmouse; 2OMeAO-treated mdx muscle (—) and senseoligonucleotide–injected control (—) were compared with age-matchedC57 muscle (—). (c) Section of 2OMeAO-treated muscle from b stainedfor dystrophin. Nuclei were counterstained with DAPI (blue).

a b

Figure 6 Dystrophin re-expression and lack of immune response. (a) Topleft, dystrophin-positive fibers 3 weeks after readministration of 2OMeAO, 3 months after initial injection. Top right, higher magnification of areashown as inset in top left panel, counterstained with DAPI. Bottom left, a single CD4+ lymphocyte (arrow) in a section adjacent to that in the topright panel. Bottom right, section adjacent to that in top right panel, showing a few mononucleated cells (arrows). Arrowheads identify bloodvessels. (b) Absence of antibody against dystrophin 3 weeks after first(serum 1) or second (sera 2 and 3) injections of 2OMeAO (AO). Sera wereapplied to sections of C57 tibialis anterior muscle. NCL-DYS1 (Dys1) is used as positive control.

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3 weeks after the second administration was even higher than after asingle injection, ranging from 589 to 790 with an average of 685 permuscle (n = 4), implying an undiminished efficiency in dystrophininduction and no significant immune complications.

We also looked for specific humoral immune responses to dys-trophin in serum from mice 3 weeks after single 2OMeAO injectionand after readministration. Using a sandwich ELISA to detect anti-bodies specific to dystrophin, we found only the same backgroundsignals in the 2OMeAO-injected mice as in both normal C57 anduntreated mdx mice. Because antigen recognition by antibodiesmay depend on the conformation of dystrophin, we also appliedthe sera as primary antibodies to sections of snap-frozen normalmuscle and used a fluorescent-labeled antibody to mouseimmunoglobulins to look for characteristic dystrophin immunos-taining. In contrast to the positive control of a known mouse anti-body to dystrophin, no membrane staining of muscle was observedwith any sera from 2OMeAO-injected mice expressing high levelsof dystrophin (Fig. 6b).

DISCUSSIONTherapeutic strategies for replacing the protein-coding region ofthe dystrophin gene by grafting normal myogenic cells, or by deliv-ering copies of functional genes through viral or nonviral vectors,have been stymied by the lack of effective distribution beyond thelocal injection site13. This raises interest in approaches that usesmall molecules that might be delivered systemically, such aschimeric DNA or RNA molecules to repair point mutations, orantibiotics such as gentamycin to allow read-through of nonsensemutations14,15. The strategy presented here, which involves redirect-ing splicing to exclude exons carrying mutations that prevent trans-lation, also uses small molecules with the potential for systemicdelivery. Its greatest appeal is that, in principle, it is applicable to themajority of DMD mutations and many other common mutationsthat could be rectified by skipping of a few particular exons10. SevereBMD patients might also benefit from skipping of exons to producea more functional dystrophin. If it proves possible to skip multipleexons, then a transcript corresponding to the minidystrophin dele-tion of exons 17–49 would represent a common optimum16.

Immunological consequences should be minimized by this strat-egy, as all sequences expressed are from the patient’s own genome.Only peptide sequences encoded at the new splice junctionsbetween previously noncontiguous exons would be novel, andmany of these would appear in ‘revertant’ fibers found in manypatients. There is also evidence of generalized, low-level produc-tion of aberrant splice isoforms of the dystrophin transcript17,18,some of which will retain the normal reading frame for peptidesthat could tolerize the individual to both exonic and aberrant splicejunction sequences. Our data are in agreement with this concept,showing no evidence of an autoimmune response to the 2OMeAO-induced dystrophin.

Following the pioneering work using antisense ribonucleotides tobias RNA splicing away from a cryptic site in mutated β-globin pre-mRNA19, efforts have been made to extrapolate its use for treatmentof DMD8–10,20. Experiments using myogenic cell cultures havedemonstrated the principle that sequence-specific 2OMeAOs caninduce targeted exon skipping to re-establish the dystrophin mRNAreading frame. However, restoration of dystrophin expression by2OMeAOs in muscle in vivo has been too low to be of practical use.In our previous study, for example, membrane-localized dystrophinin muscle in vivo was equivocal despite demonstrable induction oftargeted exon skipping in cultured cells8.

A major factor in achieving success in vivo is probably the deliv-ery of sufficient 2OMeAO to the muscle fiber nucleus to apprecia-bly skew the splicing toward the preferred transcript. Here we haveused a more precisely targeted 2OMeAO, an order of magnitudemore effective than that used previously, and administered at ahigher dose8,21. Induction of dystrophin expression remains ineffi-cient, however, when the same amount of 2OMeAO is injectedalone or with lipid-based DNA delivery agents. Expression of near-normal levels of dystrophin was reliably achieved only when2OMeAO was delivered with the help of the nonionic block copoly-mer F127, listed in the pharmacopoeia as an ‘inactive excipient’ andwidely used for drug delivery. We have found this to be the mosteffective of the block copolymers for improving plasmid deliveryinto muscle11. Here we report the first use of a block copolymer forobtaining efficient delivery of antisense oligonucleotides. F127causes no microscopic damage outside the needle track on intra-muscular injection and, for delivery of small nucleic acids intomuscle, it seems better suited than any other known reagents ordelivery systems, such as electroporation. Although not as efficientas recombinant adenovirus and adeno-associated virus vectors, andmarginally less efficient than electroporation for large plasmids, itis simple, cheap and reliable, and does not have some of the limita-tions of viral vectors, nor does it carry the dangers of electropora-tion on the human scale. As a practical therapy, antisenseoligonucleotides have the problem of a limited duration of effectand would thus have to be administered at regular intervals. Thiscould not be contemplated in muscles that are difficult to access,and routine multiple injections into any muscle would be bestavoided, so clinical trials should await further improvements inefficiency that would permit systemic vascular delivery. In thisrespect, the current clinical usage of block copolymers, togetherwith their lack of pathogenic effect in our experiments, is a greatadvantage for development of antisense oligonucleotide–basedtreatments. Their known chemical composition permits a system-atic investigation of their mode of action as a basis for rationaldesign of improved variants that might achieve vascular delivery.Development of such low-toxicity compounds for intracellulardelivery of nucleic acids has so far been limited but might prove tobe more rewarding, in terms of return for outlay, than developmentof other delivery systems such as viral vectors22.

METHODSAnimals, oligoribonucleotides and delivery methods. Three age groups ofmdx mice were used: 2 weeks, 4–6 weeks (designated 4 weeks) and 6months. Antisense oligoribonucleotides against the boundary sequences ofexon and intron 23 of the dystrophin gene (M23D(+02–18); 5′-GGC-CAAACCUCGGCUUACCU-3′ ; designated 2OMeAO) and the controlsense oligonucleotide (5′-AGGUAAGCCGAGGUUGGCC-3′) were used.Both oligonucleotides were 2-O-methylated and phosphothioated21.Oligonucleotides (5 µg; synthesized in S.D.W’s laboratory) were dissolvedin 30 µl saline with or without the block copolymer F127 (Sigma), at a finalconcentration of 300 µg/ml. We injected this mixture into each tibialis ante-rior muscle (10 µl for 2-week-old mice, 30 µl for adult mice), using thesense oligonucleotide or saline as controls in contralateral muscles.Experiments were done under animal license 70/5177, Great Britain. Micewere killed at selected time points, and muscles were snap-frozen in liquidnitrogen–cooled isopentane and stored at –80 °C.

Antibodies and immunofluorescence. Transverse sections cut at 100-µmintervals over two-thirds of the muscle length were examined for dys-trophin expression by immunohistochemistry. Intervening sections werecollected for western blotting. We also examined 7-µm serial sections with apanel of polyclonal and monoclonal antibodies for epitope mapping of

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dystrophin and detection of dystrophin-associated proteins12. Rabbit poly-clonal antibodies to the nNOS N terminus (Santa Cruz) and monoclonalantibodies to β-dystroglycan and to α- and β-sarcoglycan were usedaccording to the manufacturer’s instructions (NovoCastra). Polyclonalantibodies were detected by Alexa 594–conjugated goat antibody to rabbitimmunoglobulin (Molecular Probes); monoclonal antibodies were detectedby biotinylated rabbit antibodies to mouse immunoglobulins (DAKO) fol-lowed by streptavidin–Alexa 594 (Molecular Probes). Endogenous mouseimmunoglobulins were blocked as described previously23. T cells were iden-tified by biotinylated rat antibodies to mouse CD4 and CD8, andmacrophage and monocyte populations by a specific rat antibody24.

Western blot. Intervening sections collected in a 1.5-ml microfuge tube ondry ice were ground to powder, which was then lysed with 200 µl proteinextraction buffer8, boiled for 5 min and centrifuged. We measured proteinconcentrations of the supernatants with a Protein Assay Kit (Bio-Rad LifeScience). Protein (100 µg) was loaded onto 6% polyacrylamide gels, elec-trophoresed and blotted onto a nitrocellulose membrane that was probedwith (NCL-DYS1; a mouse monoclonal antibody to the spectrin-like roddomain of dystrophin 1:50; NovoCastra) overnight. Bound primary anti-body was detected with horseradish peroxidase (HRP)-conjugated rabbitantibody to mouse immunoglobulins and the ECL Western BlottingAnalysis System (Amersham Biosciences). The intensity of the bandsobtained from 2OMeAO-injected mdx muscles was measured and com-pared with that from normal C57 muscles.

Muscle physiology. We dissected pairs of 2OMeAO-injected and controltibialis anterior muscles from mdx mice 3–4 weeks after injection, and com-pared their contractile properties with those of normal C57 tibialis anteriormuscles by a standard method25 in Krebs-Henseleit solution at 25 °C. Eachmuscle was stimulated with a pair of platinum electrodes using a dual stim-ulator (Digitimer, MultiStim System-D330). A program written inTestPoint (Keithley Instruments) was used to control stimulation andmotor arm movement and to record force, length and stimulation. Muscleswere stimulated every 4 min to produce a fused isometric tetanus (60–70 Hz) with a duration of 0.44 s. The maximum tetanic force (P0) at theoptimum length (L0) was measured. Fatigue was introduced by repeatedtetanic stimulation every 2 s for 4 min. After the experiment, fiber length atL0 was measured under a dissecting microscope. The muscle was blottedand weighed, and CSA was calculated assuming a muscle density of1.06 mg/mm3 (ref. 26).

Detection of antibody against dystrophin. Sera taken 3 weeks after singleinjection, or 3 weeks after the second 2OMeAO injection, were comparedwith sera from normal C57 and untreated mdx mice. ELISA plates werecoated with dystrophin-specific rabbit polyclonal antibody P6 (1:2,000)overnight, washed, blocked with normal goat serum and incubated withprotein lysate from normal muscle of C57 mice for 1 h. We incubated theplates for 1 h with serial dilutions of sera from the experimental and controlmice, and washed them before incubation with HRP-conjugated goat anti-body to mouse immunoglobulins for 1 h.

To detect nondenatured dystrophin, we used serial sections of snap-frozen C57 muscle. Endogenous mouse immunoglobulins were blocked bythe method described previously23. Autoimmune antibodies in the serum(1:2 dilution) against other mouse cellular proteins were depleted by incu-bation with mouse liver powder for 1 h at room temperature. Monoclonalantibody NCL-DYS1 (1:10 dilution) was similarly absorbed. We incubatedthe blocked sections with preabsorbed serum for 1 h and then with HRP-labeled goat antibody to mouse immunoglobulins for 1 h. Sections werewashed, scraped off the slides and collected into ELISA plates. The enzymeactivity was measured by Spectramax (Molecular Devices). Duplicates ofthe sections were also probed with Alexa 594–conjugated goat antibody tomouse immunoglobulins for immunofluorescent examination.

ACKNOWLEDGMENTSThis work was supported by the Medical Research Council of Great Britain, theMuscular Dystrophy Group of Great Britain, the Leopold Muller Foundation,the Muscular Dystrophy Association of the USA and Western Australia, the

Neuromuscular Foundation of Western Australia, and the National Health &Medical Research Council of Australia. We also thank R.C. Woledge (Institute ofHuman Performance, University College London) and A. Wilson (Structure andMotion Laboratory, Royal Veterinary College, London) for providing equipmentfor part of the muscle contraction tests.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 11 April; accepted 10 June 2003Published online 6 July 2003; corrected 20 July 2003 (details online);doi:10.1038/nm897

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