implications for gene therapy of Duchenne muscular dystrophy - Nature

Gene Therapy (2000) 7, 1439–1446  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt

INHERITED DISEASE

RESEARCH ARTICLE

Immune responses to dystrophin: implications for gene therapy of Duchenne muscular dystrophy A Ferrer, KE Wells and DJ Wells Gene Targeting Unit, Department of Neuromuscular Diseases, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Hospital, St Dunstan’s Road, London W6 8RP, UK

Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse fulllength or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be

tolerant due to the presence of rare ‘revertant’ dystrophinpositive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of ‘revertant’ dystrophin-positive muscle fibres. Gene Therapy (2000) 7, 1439–1446.

Keywords: CD8; dystrophin; mdx; immunology; tolerance; Duchenne

Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy that affects one in 3500 male births and which is caused by mutations in the dystrophin gene. The severe phenotype observed for patients with DMD is due to complete absence or substantial reduction of the protein dystrophin. DMD mutations alter the normal open reading frame causing premature termination of translation which gives rise to a short and unstable protein.1 However, in Becker muscular dystrophy (BMD), an in frame deletion allows the translation of a truncated but stable product that can perform some of the functions of the full-length protein leading to a milder phenotype of the disease.2 Importantly, DMD presents with a high number of sporadic cases, varying from one third to two thirds according to different reports,3,4 and so cannot be controlled by genetic counselling alone. Possible therapeutic approaches include transplantation of healthy myoblast stem cells5 or in vivo gene transfer of a functional dystrophin gene.6–9 In DMD patients, dystrophin is either absent, present at low levels or in an abnormal form. Therefore, gene transfer of dystrophin may lead to the development of immune responses to previously unseen epitopes. This may also be true of Becker patients where specific epitopes will be missing in the truncated dystrophin that is present. The potential for immune responses to trans-

Correspondence: DJ Wells Received 9 March 2000; accepted 18 May 2000

ferred dystrophin is suggested by the presence of antibodies specific to the donor dystrophin after cardiac transplantation in a patient with BMD10 and DMD patients receiving donor myoblast transplantation.11,12 Similarly, anti-dystrophin antibodies have been noted in animal studies following congenic normal myoblast transplantation into dystrophic mice13,14 or in vivo gene delivery using recombinant adenoviruses.8,15 However, in all the above studies, other potentially foreign proteins were introduced at the same time as dystrophin and thus render difficult a complete assessment of the specific immune response to dystrophin. We have assessed specific immune responses to dystrophin using injection of naked plasmid DNA directly into skeletal muscle of the dystrophin-deficient mdx mouse. This method of nonviral gene delivery avoids the cointroduction of potential neoantigens and so allows detailed examination of the specific immune response to dystrophin. We show a strong cytotoxic response to human dystrophin thus demonstrating a potential problem associated with introduction of dystrophin into DMD patients. In contrast, despite being dystrophin deficient, mdx mice are tolerant to delivery of mouse dystrophin. This tolerance in mice suggests that careful patient selection may avoid immune responses in DMD clinical trials.

Results Multiple injections of plasmid DNA encoding dystrophin leads to an increased number of dystrophin-positive fibres in mdx-nude mice We examined the additive effects of multiple injections of plasmid DNA encoding human minidystrophin (CBC)

Immune responses to dystrophin gene transfer A Ferrer et al

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Table 1 Number of revertant fibres detected in transverse sections of tibialis anterior (TA) muscle of mdx and mdx-nude mice injected with the control plasmid CMVO compared with the contralateral uninjected TA Mice Mdx Mdx/nude

Injected 22.1 ± 3.1 19.5 ± 2.3

Uninjected 17.6 ± 2.4 14.25 ± 2.2

Figure 1 Number of dystrophin-positive fibres in transverse sections of tibialis anterior muscles from mdx and mdx-nude mice 4 weeks after the first dose of plasmid encoding human minidystrophin driven by the CMV promoter (CBC) or the HSA promoter (ABC). Each bar diagram represents the mean of six mice ± s.e.m. (쮿, injected leg; 쏔, uninjected contralateral leg).

into mdx-nude mice. Three injections produced a significant increase in the number of dystrophin-positive fibres, approximately four-fold, when compared with a single injection (data not shown). As a control, other mice were injected with three doses of the control plasmid CMV0 which did not encode dystrophin. There was no significant increase in the number of dystrophin-positive revertant fibres after three injections of plasmid in either mdx or mdx-nude muscles compared with the uninjected contralateral muscles (Table 1). As single plasmid injections sometimes failed to generate a significant number of dystrophin-positive fibres above the background level of endogenous dystrophin-positive revertant fibres, the subsequent experiments examining immune responses to dystrophin used three injections of plasmid DNA.

Human dystrophin expression stimulates both humoral and cytotoxic immune responses Two weeks after the last of three doses (4 week timepoint) of human minidystrophin driven either by the CMV intermediate–early promoter and enhancer (CBC) or the ␣-skeletal actin promoter (ABC) there was a marked difference between mdx and the T cell-deficient mdx-nude mice. With both plasmids there were significantly lower numbers of dystrophin-positive fibres in the mdx compared with the mdx-nude mice (Figure 1). Muscle sections from mdx mice also showed focal accumulations of CD8+ve cells around isolated dystrophin-positive fibres in the injected (Figure 2a and b) but not in the control leg (Figure 2c and d). Serum from the treated mdx mice showed the production of dystrophin-specific antibodies by Western blot analysis. No dystrophin-specific antibodies were present in control sera (Figure 3). An ELISA was developed and used in order to quantify difGene Therapy

Figure 2 Transverse serial sections of tibialis anterior muscle from mdx mice immunostained for dystrophin (a, c, e) or CD8+ve cells (b, d, f). Tibialis anterior (TA) muscle injected either with human minidystrophin (a, b) or with full-length human dystrophin (e, f) encoding plasmid, shows a specific CD8+ve cell response against the human epitopes. Contralateral uninjected TA (c, d) shows a dystrophin revertant fibre and the absence of CD8+ve cells. Scale bars indicate 25 ␮m.

Figure 3 Western blot analysis of specific IgG antibody titre against dystrophin. The different lanes contain muscle homogenate from transgenic mice (AD17) expressing full-length human dystrophin (1, 4 and 7), from C57Bl10 mice (2, 5 and 8) and from mdx mice (3, 6 and 9).The blots were stained using the monoclonal antibody 6C5 that recognizes the carboxyl terminus of human and mouse dystrophin (1, 2 and 3), serum from mdx mice injected with the control (CMVO) plasmid (7, 8 and 9) or serum from mdx mice injected with human minidystrophin encoding plasmid (4, 5 and 6). The band of approximately 427 kDa that corresponds to dystrophin appears in the monoclonal antibody-positive control (1) and in the immunoreactive serum (4).

ferences in the antibody titre between mdx mice injected with the human minidystrophin driven from a viral promoter (CBC) and mice injected with a plasmid in which human minidystrophin was driven from a muscle-specific eukaryotic promoter. Although there was considerable animal to animal variation, animals injected with the CBC plasmid produced higher titres of dystrophin-specific IgG2a compared with those injected with the ABC


Table 2 Titre of IgG2a antibodies specific to human minidystrophin present in the serum of mdx mice injected either with the CBC or ABC plasmid Plasmid

IgG2a

CBC mdx mdx mdx mdx mdx mdx

1 2 3 4 5 6

⬍3200 ⬍1600 ⬍10 ⬎3200 ⬎3200 ⬎3200

ABC mdx mdx mdx mdx mdx mdx

1 2 3 4 5 6

⬍3200 ⬍640 ⬍10 ⬍640 ⬍3200 ⬍640

Titres are recorded as the inverse of the serum end-point dilution. Serum from mdx-nude mice had a titre of ⬍10.

Gene transfer of murine dystrophin does not evoke an immune response Similar experiments were performed with plasmids encoding either mouse minidystrophin (CVBA) or full-length dystrophin (CVAA). Samples were taken at the 4 week and 12 week time-points. No dystrophin-specific humoral immune response was detected with either plasmid at either time-point. Likewise, there were no significant differences between the number of positive fibres in the injected legs of mdx and mdx-nude mice with either plasmid at either time-point (Figure 5a and b). Furthermore, in all cases, the number of positive fibres in the injected legs was greater that the number in the control uninjected contralateral muscle. The increased number of dystrophin-positive fibres in the CVAA injected muscles at 12 weeks was accompanied by an increase in the number of dystrophin-positive revertant fibres in the contralateral control muscles. There were some CD8+ve cells present in the mdx muscles at both 4 and 12 week time-points with both plasmids. However, in no case did we observe clustering of CD8+ve cells around individual dystrophinpositive muscle fibres.

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Discussion plasmid (Table 2). This correlated well with the significantly greater number of dystrophin-positive fibres observed in the mdx-nude animals injected with CBC compared with ABC (Figure 1). At the 4 week time-point after injection of human fulllength plasmid (CDF) there was also evidence of focal accumulations of CD8+ve cells around dystrophin-positive muscle fibres (Figure 2e and f) and the production of dystrophin-specific antibodies in mdx mice. However, there was no significant difference in the number of dystrophin-positive fibres between the mdx and mdx-nude mice (data not shown). Consequently, the experiment was repeated but samples were collected 6 weeks after the last of three doses (8 week time-point). In this case, there was a significant difference between the mdx and mdx-nude mice with the former showing no difference in the number of dystrophin-positive fibres compared with the contralateral control limb (Figure 4).

The dystrophin gene covers in the order of 3.0 mega bases and is one of the largest known genes. Dystrophin is expressed in all skeletal muscles as a large 427 kDa protein. However, the gene also produces multiple other dystrophin isoforms, which arise from a variety of tissuespecific promoters and alternative splicing events. Alternative splicing of the first exon and expression from the cortical promoter or the Purkinje promoter produces the full-length (427 kDa) brain isoforms.16,17 Other promoters

Figure 4 Number of dystrophin-positive fibres in transverse sections of tibialis anterior muscles of mdx and mdx-nude mice 8 weeks after the first dose of plasmid encoding full-length human dystrophin driven by the CMV promoter (CDF). Each bar represents the mean of seven mice ± s.e.m (쐽, injected leg; 쏔, uninjected leg).

Figure 5 Number of dystrophin-positive fibres in transverse sections of tibialis anterior muscles of mdx and mdx-nude mice 4 and 12 weeks after the first dose of plasmid encoding mouse full-length (a) or minidystrophin (b), both driven by the MCK promoter. Each bar represents the mean of six mice ± s.e.m (쐽, injected leg; 쏔, uninjected leg). Gene Therapy


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exist within the introns further downstream. These promoters give rise to tissue-specific truncated isoforms such as DP40 and DP71 (all tissues except muscle), DP116 (Schwann cells), DP140 (central nervous tissue and kidney) and DP260 (retinal). The different isoforms are reviewed by Bakker and Van Ommen.18 A further promoter 600 kb upstream of all the other promoters drives expression in lymphocytes extending the gene to three million base pairs.19 Additional complexity is added by the numerous alternative splice variants found at the carboxy terminus of the muscle isoform. At least 18 splice variants have been described for this region (reviewed by Winder20). The site of mutation will determine which isoforms (if any) are expressed in each DMD patient. The mdx mutant mouse is a naturally occurring animal model for Duchenne muscular dystrophy.21 The mdx has a point mutation (C to T) at position 3185. This base change results in a premature stop codon22 leading to a failure to express the 427 kDa isoforms of dystrophin. However, the mdx does express many of the other isoforms of dystrophin from more 3⬘ promoters. Additionally, rare dystrophin-positive ‘revertant’ muscle fibres are found within the dystrophic muscles.23 Revertants arise from different splice events during RNA processing which give rise to recombinant dystrophins of a variety of different sizes.24,25 DMD patients have a wide range of mutations. Large deletions and duplications are detected in approximately 65% of cases, another 18% are non-sense mutations, 8% small deletions and insertions, 7% splice site mutations, and 2% are missense mutations.26 Up to 50% of DMD patients show between 0.2 and 4% dystrophin-positive revertant fibres.4,27 Indeed, the presence of these spliced messages can account for patient phenotypes where these do not match that predicted from their genotyped mutation.28 The initial trials of myoblast transfer into DMD patients were unsuccessful although they did demonstrate the transient restoration of dystrophin-positive muscle fibres in the injected DMD muscle. The short-term dystrophin expression may have been partly due to immunological rejection as supported by the detection in some patients of serum antibodies against dystrophin and a range of other proteins after myoblast transfer.12 In the present study, only transient expression of the human dystrophin was observed in immunocompetent mdx mice when plasmid encoding for human minidystrophin was injected. However, a high level of expression was maintained in the immunodeficient mdx-nude mice. Four weeks after the first injection the number of fibres expressing dystrophin in the injected leg of mdx mice was not significantly different from the basal level of revertant fibres indicating that the expression due to the gene transfer had been largely eliminated. This decline in the number of dystrophin-positive fibres in the immunocompetent mdx might be attributed to the inactivation of the promoter over time or the toxicity of the protein. However, these mechanisms also would had affected the expression in nude mice and this was not the case. Interestingly, overexpression of dystrophin in transgenic mdx mice does not appear to cause any toxicity problems.29 The most likely explanation for the decline in dystrophin expression is the destruction of the dystrophintransfected fibres. This destruction of muscle fibres could have been due to a cell-mediated immune response

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through cytotoxic CD8+ T cells and this is strongly suggested by the clustering of CD8+ cells around dystrophinpositive fibres in the injected leg. This appeared to be a human dystrophin-specific cytotoxic response as no such clustering was observed around the revertant mouse dystrophin-positive fibres in the contralateral control muscles. Intramuscular injection of plasmid encoding ␤galactosidase generated a similar cytotoxic response that led to destruction of transfected muscle fibres in normal but not in nude Balb/c mice.30 A decline in the number of fibres in the mdx compared with nude mdx was also observed when the muscle-specific skeletal actin (HSA) promoter was used to drive expression of minidystrophin. However, the drop in dystrophin-positive fibres was not as dramatic as the results in animals treated with the CMV-driven minidystrophin plasmid. This may be a reflection of the lower maximum level of expression observed with this eukaryotic promoter both in total expression and the number of transgene-positive fibres.31 The latter effect is shown by the total number of dystrophin-positive fibres in the mdxnudes injected with these two constructs. It is unlikely that muscle fibres themselves act as antigen-presenting cells as they exhibit only low levels of MHC I32 and do not appear to possess costimulatory molecules such as B7.1 or B7.2. It is more likely that dendritic cells or macrophages present in the muscle are directly transfected with the plasmid DNA and present dystrophin in the context of MHC I. If professional antigenpresenting cells are not directly transfected, they may receive antigen released from damaged or leaky transfected myofibres and present peptides on MHC I via a cross-priming pathway.33 The immune response to dystrophin expressed by a muscle-specific promoter suggests that the cross-priming pathway may be as important or more important than direct transfection of dendritic cells in triggering the initial immune response, although some ‘leaky’ expression from this muscle-specific promoter in dendritic cells cannot be ruled out. Loirat and colleagues34 have reported similar immune responses to hepatitis B virus envelope proteins when expressed from a muscle-specific promoter or the viral CMV promoter. Plasmid transfer of human full-length dystrophin generated a cytotoxic immune response similar to that generated with human minidystrophin plasmid injections. However, the response was more delayed with the fulllength plasmid with no significant decline in full-length dystrophin-positive fibres at 4 weeks in contrast to a rapid loss of minidystrophin-positive fibres over the same period. This may again be a simple reflection of the level of transgene expression as more positive fibres were detected in mdx-nude mice after minidystrophin plasmid injections compared with full-length plasmid. Alternatively, it might be that the full-length dystrophin is less immunogenic than the minidystrophin, possibly due to suppressor epitopes in the rod domain (deleted in the minidystrophin). In contrast to the results with human full or minidystrophin, no decline in the number of dystrophin-expressing fibres in the immunocompetent mdx mice was observed at 1 or 3 months when either mouse full or minidystrophin was injected. The mdx mouse seems to be tolerant to mouse dystrophin. This result correlates with the report of successful mouse myoblast transplant into mdx despite the detection of antibodies against dystrophin,13,14


although others have recently reported the rejection of normal apparently isogenic myoblasts by mdx mice via a CTL response.35 As previously mentioned, the plasmid gene transfer system does not involve other foreign proteins whereas cells may present non-familiar proteins following strain divergence or as a contaminant from serum used in cell culture. Similarly, CTL responses acting against muscle fibres following adenovirus-mediated dystrophin gene transfer could be directed to proteins associated with the viral vector. Such immune responses to foreign proteins may alter the nature of immune responses to dystrophin. Although both human and mouse dystrophin molecules are 90% identical, some epitopes are clearly sufficiently different to trigger an immune response to human dystrophin in mice. As most preclinical studies of dystrophin gene transfer have been conducted using human dystrophin constructs, some trials may have seriously underestimated the efficiency of different approaches due to destruction of transfected fibres following immune responses to human dystrophin. The presence of dystrophin-positive revertant fibres in mouse muscle could explain the lack of rejection of fibres transfected with mouse dystrophin constructs. Such fibres are likely to be the only source of the amino terminus and the first half of the rod domain of dystrophin found in the full-length skeletal muscle isoform. It is possible that other smaller isoforms, unaffected by the stop mutation in the mdx, also play a role in this tolerance to full-length dystrophin. In either case, these observations suggest that it will be important to assess the precise genotype and phenotype in Duchenne patients presented for gene therapy trials. Patients with small mutations and some revertant fibres are unlikely to show problems with immune rejection of transfected muscle fibres following gene therapy. However, those patients with no detectable revertants or with large deletions may not be tolerant to all newly presented epitopes unless they are subjected to a regimen of immunosuppression.

Materials and methods Constructs The SV40 t splice and polyadenylation signals from pMSGCAT were subcloned into the XhoI and ApaI (repaired) sites in pBluescript SK+ (Stratagene, Cambridge, UK) making pSKpA. The cytomegalovirus immediate early–promoter (CMV) was obtained as an EcoRI (repaired) to NotI restriction fragment (which includes a splice donor pair from SV40) from CMV␤ (Clontech, Palo Alto, CA, USA). This fragment was inserted between the NotI and SacI (repaired) sites in the pSKpA polylinker producing CMVpA. A NotI/SalI fragment from RSVDy or RVSDyB encoding full-length human dystrophin or human minidystrophin respectively6 (cDNAs from George Dickson and Kay Davies (University of Oxford, Oxford, UK), respectively) were directionally cloned into the same sites in CMVpA to produce CBM and CDF. The muscle promoter-driven human minidystrophin transgenic construct, AB, has been described previously.36 Expression is driven by the HSA promoter (−2200 to +2 from HSA2000CAT37). The plasmid construct, ABC, is a modification of this transgenic construct in which the AsuII to SalI 3⬘ terminal fragment

from plasmid K1 (Don Love, University of Auckland, Auckland, New Zealand) was used to replace the same restriction fragment in the AB construct thus exchanging the 285 base pair (bp) 3⬘UTR in the transgenic construct with the complete 3⬘UTR of 2.7 kb (11242–13949, Accession No. 18533). The AsuII/SalI fragments encoding the carboxy terminus region and containing the complete 3⬘UTR sequences were moved from ABC (2.7 kb 3⬘UTR) into the same sites in CBM replacing the 267 bp 3⬘UTR (11242–11527) with the complete 3⬘UTR kb producing CBC. A further CMV promoter-driven full-length human dystrophin construct on a different plasmid backbone and containing only 871bp (11242–12112) of 3⬘UTR was supplied by Serg Braun, Transgene, France. This plasmid is described in more detail in the accompanying paper by Braun et al (this issue, pp 1447–1457). Murine full-length and minidystrophin constructs CVAA and CVBA were supplied by Jeff Chamberlain (University of Michigan, Ann Arbor, MI, USA). Expression is driven by the 3.3 kb muscle creatine kinase promoter. These constructs contain the VP1 intron from SV40 3⬘ to the promoter and also contain the complete 3⬘UTR of dystrophin (Table 3). These constructs have been previously described.38

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Preparation of plasmid DNA Plasmids were grown in XL1Blue (Stratagene, Cambridge, UK) and purified using ENDOfree plasmid kits from Qiagen (Crawley, UK) following the supplier’s protocol. Identity was confirmed by agarose gel electrophoresis of both uncut and restriction digested plasmids. Contamination with RNA was not observed and the majority of each plasmid was present as covalently closed circles. Plasmid preparations were analysed for endotoxin (lipopolysaccharide) contamination using Pyrogent 500 turbimetric assay (BioWhittaker, Wokingham, UK) DNA. All plasmid preparations used in this study had LPS contamination ⬎0.05 EU/␮g. Intramuscular injection of plasmid DNA In vivo experiments were carried out on 7- to 10-weekold mice. Animals were housed in a minimal disease facility with food and water ad libitum. Plasmid DNA (2 ␮g/␮l) in normal saline was injected percutaneously. All injections (25 ␮l) were carried out using a 27-gauge needle in a proximal to distal direction inside the anterior tibial muscle, under fentanyl/fluanisone and midazolam general anaesthesia (respectively, Hypnorm; Janssen Pharmaceutical, High Wycombe, UK and Hypnovel; Roche, Welwyn Garden City, UK) as previously described.30 Three injections of plasmid were performed on the same muscle with intervals of 1 week between injections. Samples were then collected 2 to 10 weeks after the last injection. Table 3 Dystrophin plasmids Construct CBC ABC CVBA CDF CVAA

Promoter CMVIE HSA 2.2 kb MCK 3.3 kb CMVIE MCK 3.3 kb

Coding region minidystrophin minidystrophin minidystrophin full-length dystrophin full-length dystrophin

Species human human mouse human mouse

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Collection of samples Mice were killed by injection of 0.1 ml pentobarbital (Euthetal; Rhone Merieux, Harlow, UK) and blood was collected via cardiac puncture. Both TAs were excised post mortem together with a piece of liver and kidney as a marker for the injected and control TA. Tissues were mounted on a cork block, embedded in Cryo-M-Bed (Bright, Huntingdon, UK) and snap-frozen in liquid nitrogen-cooled isopentane. The frozen muscles were stored at −70°C. The blood samples were left for a few hours at 4°C for the blood clot to retract and were spun twice at 9000 g for 5 min. The serum was aliquoted and stored at −70°C until used in the Western and ELISA assays. For analysis of transfected fibre number following in vivo experiments, 10 ␮m cryostat sections were cut at a minimum of 10 evenly spaced levels throughout each muscle and lifted on to APES-coated slides. Slides were stored at −70°C after air drying. Production of polyclonal antibody to dystrophin A multiple antigenic peptide (MAP) of the last 17 amino acids of dystrophin was used to immunise rabbits. The resulting sera were peptide specific on ELISA and the best sera (CR2) were purified over a Mabtrap column (Pharmacia, St Albans, UK) according to the manufacturer’s instructions. This antibody recognised a 427 kDa band on Western blot and only stained ‘revertant’ fibres in cryosections from mdx muscle. ELISA assay for specific antibodies to dystrophin A sandwich quantitative ELISA assay for antibodies to dystrophin following gene therapy was based on a method of Ishikawa et al.39 Greiner (Stonehouse, UK) Hybind ELISA plates were coated overnight at 4°C with a 1:50 dilution of sheep anti-mouse IgG1 in TBS buffer using 50 ␮l per well. The plates were then washed four times with TBST and blocked by incubating the plate with a 3% solution of dried milk (Marvel; Premier Beverages, Knighton, UK) in TBST for 30 min at room temperature. Plates were then incubated for 1 h at 37°C with 50 ␮l per well of a 1:10 dilution of 6C5 monoclonal IgG1 antibody to the carboxyl terminus of human dystrophin (cross-reacts with mouse dystrophin, kindly supplied by Louise Anderson, University of Newcastle, Newcastle upon Tyne, UK).40 As a source of dystrophin, a muscle homogenate from the transgenic line AD17 overexpressing human dystrophin was employed and a similar homogenate from mdx mice acted as the background control. Muscle homogenates were prepared as for Western blotting at an approximate concentration of 100 mg/ml. They were diluted to 1:50 in TBS, 1% Triton, 5% FCS pH 8 and 100 ␮l were incubated per well for 1 h at 37°C (200 ␮g per well was sufficient to saturate the binding capacity of the catching antibody 6C5). Bound dystrophin was detected with either the 6D3 monoclonal IgG2a antibody to the central rod domain4 or the serum from injected mice. A series of two-fold dilutions starting from 1:10 of each animal serum were prepared in TBST and added to the wells containing the AD17 or the mdx homogenate. A 10 min blocking incubation in 3% milk and 5% FCS was incorporated after the last two incubations. Finally, plates were incubated for a further 1 h at 37°C with 50 ␮l of a 1:10 000 dilution of biotinylated rabbit anti-mouse IgG2a antibody (1 mg/ml; Seralab, Bicester, UK) followed by 30 min with a 1:10 dilution of horserad-

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ish peroxidate-conjugated streptavidin/biotin complex (ABC-HRP complex; Dako, Ely, UK) in 50 mm Tris buffer pH 7.5. OPD was used to give a colourimetric quantification of the level of dystrophin in the wells that was read at 492 nm in a spectrophotometer 30 min after the OPD addition. A plot of the OD against the dilution of serum was obtained for each animal, and sera with the strongest reactivity were re-examined with doubling dilutions up to 1:3200. The titre was recorded as the inverse of the dilution that gave an OD = 0.0 once the background had been subtracted.

Immunostaining for dystrophin and CD8 cells For immunostains, several tibialis anterior muscles were mounted in a single block with small strips of either kidney or liver tissue placed adjacent to the experimental (injected) muscles to allow for correct orientation of samples after sectioning. The injected and contralateral muscles of each mouse were mounted on the same block to allow direct comparison of fibre number under the same staining conditions. Endogenous biotin background was blocked using the avidin–biotin blocking system from Dako, following the manufacturer’s instructions. Sections were stained using IgG fractionated CR2 polyclonal at a dilution of 1:200. Sections were then incubated with biotinylated anti-rabbit Ig at 1:400 (Dako), followed by horseradish peroxidase-conjugated streptavidin/ biotin complex (ABCHRP complex; Dako). Hydrogen peroxide activated DAB was used for detection. Colour development was observed microscopically and stopped at 1–3 min. Sections were washed in water, dehydrated through graded alcohols, cleared with xylene and mounted in DPX. For immunostaining of CD8+ve cells, slides were fixed in acetone for 10 min and nonspecific binding blocked as described above. The primary antibody rat anti-mouse CD8 (YTS 169) was used at a dilution of 1:2 in PBS followed by the secondary antibody, biotinylated rabbit anti-rat (Dako) at a dilution of 1:500. Detection was as described above using streptavidin–biotin complex (Dako) and DAB. Muscle sections from all levels of each muscle were examined and the section with the highest number of dystrophin-positive fibres was recorded for each muscle. A Leica DMLB research microscope (Leica, Wetzlar, Germany) with a colour JVC Ky-F55B 3-CCD camera was used to import images to a computer. Images were captured using SnapperTool version 2.04 software from DataCell Ltd, Maidenhead, UK. Western blotting for dystrophin Tibialis anterior muscles (average weight 30–40 mg) were homogenised in a final volume of 400 ␮l of SDS sample buffer (10% SDS). After SDS-PAGE, proteins were transferred on to polyvinylidene difluoride (PVDF: HybondP; Amersham, Little Chalfont, UK). Blots were air dried over night. After transfer by Western blot, the equality of transferred protein per lane was assessed by amido black stain of the membrane. Additionally, all antibodies give a background cross-reaction on over-exposure of blots and comparison of the intensity of these nonspecific bands allowed a further assessment of relative protein loading. Monoclonal 6C5 against the carboxy terminal 17 amino acids of dystrophin was used as the positive control at a dilution of 1:100. Sera from mice injected with


the various plasmids were used at a dilution of 1:10. Secondary antibody, goat anti-mouse horseradish peroxidase (HRP)-conjugated (blotting grade from Biorad, Hemel Hempstead, UK) was used at dilution of 1:5000. Antibodies were detected using enhanced chemiluminescence as directed by the manufacturer (ECL; Amersham). Blots were exposed to blue sensitive X-ray film (GRI, Braintree, UK) for a range of exposure times from 10 s to 2 min. Films were processed in an automatic X-ray processor (GRI) and were later scanned to produce images.

Statistical analysis Statistical analysis was carried out by one-way ANOVA followed by multiple paired comparison. Groups were considered significantly different when P ⬍ 0.05. Statistical tests were conducted using Sigma-Stat from Jandel Scientific (Erkraph, Germany).

Acknowledgements We wish to thank the following: David Baker (UCL, London) for the anti-CD8 antibody; Terry Partridge (MRC CSC, London) for the founding stock of our mdxnude colony; Jeff Chamberlain (Michigan) for the mouse dystrophin plasmids; Kay Davies and Don Love (Oxford) and George Dickson (Royal Holloway, London) for the human dystrophin cDNAs; Louise Anderson (Newcastle) for the anti-dystrophin monoclonal antibodies; Serge Braun (Transgene, Strasbourg) for the alternative human full-length dystrophin plasmid and Jill McMahon (Gene Targeting Unit) for advice on histology and imaging. This work was funded by the MRC, the Muscular Dystrophy Campaign and the Wellcome Trust.

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