Expression of human dystrophin following the transplantation ... - Nature

Gene Therapy (1998) 5, 1340–1346  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt

Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts P-A Moisset1, Y Gagnon1, G Karpati2 and JP Tremblay1 1

Centre de recherche en Neurobiologie, Universite´ Laval and Hoˆpital de l’Enfant-Je´sus, Que´bec; and 2Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montre´al, QC, Canada

Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppressive drugs. To determine the conditions to be used in this kind of approach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI of 0.01–1000) with an adenoviral vector containing a CMV promoter/enhancer driven 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for transgene expression. In these cultures, dystrophin mRNA was found to be proportionate with increasing MOI. Primary myoblast cultures derived from transgenic mdx mice expressing ␤-Gal under a muscle-specific promoter and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibialis muscles

of SCID mice. Ten and 24 days after transplantation, numerous muscle fibers expressing both human dystrophin and ␤-Gal were detected throughout the mouse muscles by immunohistochemistry using an antibody specific for human dystrophin. The presence of the human mini-dystrophin mRNA was also detected by RT-PCR. These results demonstrate that three essential conditions in autologous myoblast transplantation can be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle fibers; and (3) the high expression of the dystrophin transgene in situ. Furthermore, this article provides a novel RT-PCRbased technique to quantify the human dystrophin minigene expression in vitro and in vivo.

Keywords: myoblast transplantation; adenovirus infection; dystrophin minigene; Duchenne muscular dystrophy

Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which occurs in one in 3500 male births.1 It causes progressive skeletal and cardiac muscle degeneration and premature death of the patient. Two major strategies have been investigated for possible treatment of the disease: gene therapy and myoblast transplantation. The first implies direct administration of vectors capable of restoring the missing sarcolemmal protein, dystrophin, in the patient muscles. So far, firstand third-generation adenoviruses seem to be the vectors which yielded the best results for the delivery of either dystrophin minigenes or full-length cDNA.2–6 However, one of the problems which has limited the success of gene therapy with these vectors is the immune response raised against those vectors. Humoral and cellular immune reactions led to the rejection of the transduced cells and fibers, probably due to the initial adenoviral capsid load and possibly low level expression of early and late viral antigens which, in turn, are presented by the infected cells.7–10 Our research groups have shown that the use of immunosuppressive drugs such as FK506 or monoclonal antibodies against CD4, CD8 and LFA-1 can greatly reduce such reactions and permit sustained transgene

Correspondence: JP Tremblay, Centre de recherche en Neurobiologie, Hoˆpital de l’Enfant-Je´sus, 1401, 18e Rue, Que´bec (Qc), Canada G1J 1Z4 Received 25 September 1997; accepted 5 June 1998

expression after adenovirus-mediated gene therapy.11–13 Moreover, the use of a new generation adenovirus, that contains either modified or no viral genes, could greatly reduce the immune reactions against the vector-infected cells.5,6,14–16 Two significant obstacles are still present in the use of direct gene therapy. First, virus-based gene therapy leads to the infection of all cells present at the injection site, including some professional antigen-presenting cells (APC). Upon infection by first-generation viruses, these APCs are known to stimulate the humoral and cellular immune response which, in turn, damage the infected cells and reduce the effectiveness of the viral vectors.17 Again, new generation adenoviruses which have no viral antigen to present could possibly avoid this obstacle. However, a second problem is that in DMD patients older than 5 years, the absolute number and the regenerating capacity of myoblasts are greatly diminished because of repeated muscle fiber necrosis.1 Even if all the remaining myoblasts were efficiently transduced, their regenerative capacity might not be sufficient to generate enough muscle to produce an increase in strength. Adenoviral infection of mature post-mitotic muscle fibers has been thought possible for a number of years.18 However, some studies indicate that this may not always be the case.19–21 It thus seems that myoblasts are the main if not the only targets for adenovirus-based gene therapy. Transplantation of genetically modified myoblasts (‘ex vivo gene therapy’) could, however, be used successfully to bypass both problems by: (1) providing a novel myo-

Transplantation of ex vivo modified myoblasts P-A Moisset et al

blast pool containing the transgene; and (2) transplanting only myogenic cells already infected and devoid of APC. Clinical trials have proved to be of only limited success using normal donor myoblast transplantation in DMD patients, even when the donor and host were monozygotic twins.22–24 However, in dystrophic immunodeficient mice or immunosuppressed mice, monkeys, and in a few humans having an almost perfectly matched MHC, myoblast transplantation has proven successful.23,25–27 Thus, adequate immunosuppression is of prime importance to control the immune reactions against donor myoblasts. Specific immune responses are triggered not only by incompatible MHC determinants but also by minor antigens present in myoblasts and newly formed myofibers.28 Without adequate immunosuppression, these cells are thus rejected. Control of this specific immune response may require sustained immunosuppression of DMD patients. However, long-term use of immunosuppressive drugs such as FK506 and cyclosporine has been reported to provoke undesirable side-effects, such as neurotoxicity and nephrotoxicity.29 By transplanting the host’s own cells (autologous transplantation) after in vitro insertion of a functional dystrophin gene, immune rejection could be avoided, thus eliminating the need for long-term use of immunosuppressants. To circumvent the autologous myoblast depletion in DMD patients, such a procedure would have to be carried out in young patients where there are still sufficient myoblasts. The present article reports: (1) the successful transfer of mdx/␤-Gal myoblasts transduced by a first-generation adenovirus carrying a dystrophin minigene expression cassette into immunodeficient SCID mice; and (2) a novel RT-PCRbased technique to quantify human dystrophin minigene expression in vitro and in vivo.

Results

levels of mini-Dys mRNA were observed at a high MOI (between 100 and 1000), as revealed by densitometric analysis (Figure 2a and b). In these cultures, with a MOI of 1000, we found the expression of human mini-Dys to be roughly 20 times higher than mouse dystrophin. As illustrated in Figure 2a, a competitive reaction between mouse and human dystrophin products was observed with increasing MOI. None the less, in the conditions used, a linear augmentation of mouse/human dystrophin ratios versus MOI was still measured (Figure 2b). A mdx/␤-Gal muscle primary culture was infected as described in Materials and methods and left in normal culture medium until sufficient fusion was observed (roughly 3 days). In these cells, ␤-Gal is under the control of a muscle-specific promoter that is only active after myogenic differentiation. Therefore, only the myotubes or mature myoblasts are ␤-Gal+. As shown in Figure 3a and b, using a MOI of 400, equivalent fusion was obtained in both infected and control cultures, indicating that the adenoviral infection did not noticeably inhibit myoblast differentiation. The cells were then immunostained for dystrophin. As shown in Figure 3d, about 100% of mature myoblasts and myotubes are dys+, while only rare dys+ cells could be observed in the uninfected control cells (Figure 3c). Since the antibody used for this immunofluorescence technique is not human dystrophin specific, these positive cells in the uninfected controls must be revertants.

Dystrophin minigene expression in transplanted muscles The SCID muscles were transplanted with 1 × 106 or 5 × 106 infected mdx/␤-Gal cells. Ten days later, the muscles were collected and the total RNA extracted. As shown in Figure 4, expression of the human mini-Dys is only detected in muscles injected with infected cells. Moreover, a direct correlation between the initial amount

Adenovirus infection of myoblasts Different multiplicities of infection (MOI) ranging from 0.01 to 1000 were tested on cultures of mdx myoblasts. At the highest MOI, a low level of cell toxicity was detected by morphological observation. A rapid augmentation of the PCR product correlating with increasing MOI was observed from MOI 1 to MOI 1000 (Figure 1). Under the conditions used, the dystrophin minigene was not detected in non-infected cells or in cells infected with a MOI lower than 1. Dystrophin minigene expression in culture To verify the relative expression of mini-Dys mRNA, an RT-PCR was performed on the infected cells. Appreciable

Figure 1 Adenoviral infection of mdx myoblasts in culture with increasing MOI. PCR amplification of the dystrophin minigene fragment.

Figure 2 Endogenous and transgenic dystrophin expression in mdx myoblasts with increasing MOI. RT-PCR amplification of mouse and human dystrophin (a). Quantification by densitometric analysis of the RT-PCR products following EcoRV digestion (b).

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a

b

c

d

Figure 3 Dystrophin and ␤-Gal expression in infected mdx/␤-Gal myoblast cultures. Infected (b, d) and non-infected (a, c) cultures are shown after differentiation. ␤-Gal staining (a, b). Dystrophin immunostaining (c, d).

Table 1 summarizes the most relevant parameters observed in all the transplantations. Results were compiled by counting Dys+ fibers (⬎5 ␮m) in whole sections containing the most ␤-Gal+ fibers. Similar numbers of ␤Gal+ fibers were obtained following the transplantation of uninfected myoblasts (data not shown). Figure 4 Dystrophin minigene expression in the muscles of SCID mice transplanted with non-infected and infected cells. RT-PCR amplification of the dystrophin endogenous and transgenic mRNA 10 days after transplantation. Tracks: (1), 50 kb-molecular weight ladder; (2), transplanted with 1 × 106 non-infected mdx/␤-Gal cells; (3), transplanted with 1 × 106 infected mdx/␤-Gal cells; (4), transplanted with 5 × 106 non-infected mdx/␤-Gal cells; (5), transplanted with 5 × 106 infected mdx/␤-Gal cells.

of grafted cells and the expression of mini-Dys mRNA 10 days later was observed (compare lanes 3 and 5). Interestingly, we observed that, by transplanting 5 × 106 infected cells, comparable amounts of mini-Dys and normal endogenous mouse dystrophin mRNA were obtained (Figure 4). To verify the fate of transplanted myoblasts, we immunostained the muscle sections for human dystrophin and compared the results with the sections stained with X-gal 10 days (not shown) and 24 days (Figure 5) after transplantation. Good positional correlation was found between the fibers expressing human dystrophin and those expressing ␤-gal in serial sections.

Discussion The possible toxic effects of long-term use of immunosuppressive drugs such as FK506 or cyclosporine have been well documented.29 To avoid this possibility, autologous transplantation of genetically modified myoblasts Table 1 Comparative results of genetically modified myoblasts transplantation 10 and 24 days after transplantation Days after transplantation 10 24b a

␤-Gal+ fibers (% of total) 18 49 ± 3

Dys+ fibersa Dys+ fibers % of ␤-Gal+ fibers 150 188 ± 63

54 20 ± 4

Results from the sections apparently containing the most positive cells. Abundant ␤-Gal- and dystrophin-positive myotubes of diameter ⬍5 ␮m were observed but not counted (Figure 5). b Results from two different animals expressed as mean ± s.d.


has been proposed and this article describes the initial steps towards this approach. Adenoviral infections can be achieved using high virus-to-cell ratios (MOI) but high MOI have proven toxic to cultured myoblasts or to inhibit differentiation.18 Thus, a careful balance between high transgene expression and low cell toxicity must be attained. The presence and expression of the human mini-dystrophin transgene was first characterized in cultured mdx myoblasts. Efficient transduction and transgene expression without cell toxicity were observed by PCR and RT-PCR at MOI higher than 100 and lower than 1000. A MOI of 400 was therefore chosen for the infection of myoblasts before their transplantation. Ten days after transplantation, 18% of the muscle fibers were ␤-Gal+ and among these, 54% were Dys+. This result correlates more or less with dystrophin expression in cultured myoblasts/myotubes. Indeed, in culture, practically all the observed ‘mature myoblasts’ and myotubes were Dys+. These muscle sections also contained large regenerating regions in which a high number of small myotubes (⬍5 ␮m) were found. Although the exact number of these myotubes is difficult to assess because of their size and the fact that they are found in clusters, the histochemistry clearly shows that they are ␤-Gal+ myotubes. A much higher number of ␤Gal+ fibers were found throughout the grafted mouse muscles 24 days after myoblast transplantation (approximately 50% of all fibers), indicating the efficiency of the transplantation and supporting the hypothesis that the regenerating regions observed after 10 days have developed into mature ␤-Gal+ muscle fibers. A surprising result was that, although the absolute number of Dys+ fibers increased, the Dys+ cells/␤-Gal+ cell ratio was markedly lower at 24 days than at 10 days after transplantation. Furthermore, as shown previously, the Dys+ fiber/␤-Gal+ fiber ratio was also lower 10 days after transplantation than in the cultured myoblasts/ myotubes. These results can be explained by the fact that the adenovirus DNA remains episomal, without integrating with the host DNA or replicating itself, whereas the ␤-Gal gene is integrated into the genome of the myoblasts originating from a transgenic animal. When the infected regenerating myoblasts divided in situ, they progressively lost copies of the viral DNA but not of the ␤-Gal gene; thus, the expressed transgene fell below detection level. Some ␤-Gal+/Dys+ myoblasts still present in the regenerating regions of the muscle 10 days after transplantation may have divided and lost the Dys transgene before fusing and forming mature fibers during the following 14 days (ie the 24 day result). Ten and 24 days after transplantation, small diameter ␤-Gal+ and Dys+ fibers were found often close to what seems to be regenerating regions of the muscle, thus indicating recent fiber formation (shown in Figure 5). None the less, most of the small ␤-Gal+ myotubes found in these regions did not stain positively for dystrophin. These fibers have probably been formed by myoblasts which have divided many times and lost the episomal dystrophin gene. Additionally, some underestimation of the total number of Dys+ fibers might have occurred because of the restricted dystrophin nuclear domain. Indeed, our group has shown that, when a myoblast fuses to a preformed fiber and expresses both ␤-Gal and dystrophin, the former protein diffuses further from the point of its translation due to its smaller size and cytosolic localization, thus render-

ing it detectable on more serial sections of a muscle.30 For technical reasons, only one cryosection per muscle has been counted. It is therefore possible that a given ␤Gal+/Dys− fiber in a section is ␤-Gal+/Dys+ in another section that is closer to where the myoblast initially fused. Noticeably, we have obtained similar numbers of ␤-Gal+ fibers with infected and control myoblasts indicating the low infection-related death of myoblasts. In our experiments, the immunogenicity of the firstgeneration adenovirus used to infect myoblasts necessitated the transplantation of immunodeficient mice. Moreover, ␤-Gal by itself is known to be immunogenic. In immunocompetent animals, both factors would have induced an immune reaction leading to the rejection of the transduced cells.7–10 New generation adenoviral vectors that contain no viral sequences could perhaps enable us, with the same protocol, to transplant immunocompetent animals without sustained immunosuppression. Three elements would further ameliorate the procedure: (1) instead of collecting the patient’s myoblasts, one could transform other cell types into myoblasts by co-infecting both MyoD and mini-dystrophin genes; (2) since 90% of the injected cells are thought to die soon after transplantation,38,39 a treatment is needed to increase myoblast survival; and (3) as myoblasts have been shown to remain in close vicinity to the injection sites, increasing myoblast motility immediately after transplantation with appropriate additives to the culture medium could increase their migration.40 The present work also provides a new and simple way to measure the expression of the mini-Dys transgene in situ. If this procedure was to be used in future human transplantations, the infected myoblasts would have to be tested for fusion efficiency and mini-Dys expression in an animal model. This could be done in SCID mice where human myoblasts have been previously shown to fuse normally with mouse fibers.41 Until now, immunohistochemistry with a human-specific antibody has been the only method to evaluate human dystrophin expression in a mouse muscle.41

Materials and methods Cell cultures and animals Muscle biopsies were obtained from mdx/␤-Gal newborn mice (cross between C57 BL10J mdx/mdx mice (Jackson Laboratories, Bar Harbor, ME, USA) and TnILacZ mice).31 The primary myoblast cultures were obtained using a technique already described.32 The resulting cells were maintained at 37°C, with 5% CO2 in M199 medium containing 15% donor calf serum (Gibco BRL), penicillin G (10 000 IU/ml; Gibco BRL) and streptomycin (10 mg/ml; Gibco BRL) for 24 h. The immunodeficient BALB/c SCID mice (2 months old) were obtained from Jackson Laboratories. Myoblast infection The ⌬E1-⌬E3 adenoviral vector AdCMV-Dys was produced and purified as described before.4 A previously frozen adenovirus stock solution of 1010 p.f.u./ml was used to infect 5 × 106 cells at a MOI of 400. Briefly, the cells were rinsed twice in Hank’s balanced salt solution (HBSS; Gibco BRL) and were incubated with 2 ml of M199 containing the virus for 2 h at 37°C, 5%CO2. They

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a

b Figure 5 Staining of SCID muscles 24 days after transplantation. ␤-Gal staining (a). Dystrophin immunostaining (b). The solid arrowheads in the panels show the small ␤-Gal+ and dystrophin-negative myotubes observed close to regenerating regions of muscle.


were then rinsed three times in HBSS, harvested by trypsination and further processed for transplantation. Small quantities of cells were replated on LabTek slides (Gibco BRL) and incubated for 2 more days at 37°C for the immunofluorescence technique (see below).

Myoblast transplantation The left hind legs of the SCID mice were irradiated (20 Gy) 3 days before the transplantation was carried out. As described earlier, this dose of irradiation inhibits the proliferation of the host myoblasts.33,34 The day before transplantation, the left tibialis anterior muscle (TA) was surgically exposed and 7 ␮l of notexin (5 ␮g/ml) were injected into several sites in the muscle to induce fiber necrosis.35 On the day of transplantation, the infected cells (2.5 × 106 for the immunohistochemistry experiment, 1 and 5 × 106 for the RT-PCR experiment) were collected, pelleted and resuspended in approximately 15 ␮l of HBSS. This volume was then injected into multiple sites in the left TA. Cell viability was assessed by trypan blue staining. Muscle removal and preparation Ten or 24 days after transplantation, the mice were deeply anesthetized and perfused using a 0.9% sodium chloride, 2 IU/ml heparin solution. The grafted and contralateral TA were then removed and immersed overnight in a 30% sucrose solution at 4°C. Muscles were embedded in Cryomatrix compound (Shandon, Pittsburg, PA, USA), frozen in liquid nitrogen and cryosectioned at 10 and 30 ␮m. ␤-Gal histochemistry The 30 ␮m muscle sections and cells in culture were fixed with 0.25% glutaraldehyde in PBS. They were then rinsed three times with PBS. ␤-Gal expression was revealed with overnight incubation in a 0.4 mm X-gal (Boehringer Mannheim, Laval, Qc, Canada), 3 mm potassium ferrocyanide, 3 mm potassium ferricyanide, 100 ␮m magnesium chloride and PBS solution.

Dystrophin immunochemistry and immunofluorescence Human dystrophin minigene (mini-Dys) expression was detected on 10 ␮m sections by an immunoperoxidase technique using the NCLDys3 monoclonal antibody.36 The secondary antibody was a rabbit anti-mouse coupled to peroxidase (Dako, Mississauga, Ont, Canada). The peroxidase activity was revealed with diaminobenzidine (0.25 mg/ml) and hydrogen peroxide (0.015%). To detect dystrophin in myoblasts and myotubes in culture, we used a polyclonal rabbit anti-dystrophin that recognizes the C-terminal part of all the mammalian dystrophins (dilution 1:750; generous gift from Dr JS Chamberlain, University of Michigan). The secondary antibody was in that case an anti-rabbit coupled to FITC (dilution 1/40; Dako). RT-PCR detection of dystrophin minigene mRNA The expression of mini-Dys mRNA in culture and in the transplanted muscles was detected by reverse transcriptase RT-PCR. In culture, the cells were lysed and the total RNA was directly isolated in TRIZol (Gibco BRL, Burlington, Ont, Canada). For use of the muscles, the mice were deeply anesthetized and perfused briefly with the sodium chloride/heparin solution. The muscles were

then removed and immediately frozen in liquid nitrogen. Total RNA was extracted with TRIZol and treated with DNAse (Gibco BRL). A primer set was made to amplify both human and mouse dystrophin fragments under the same conditions. The 5′ primer (5′-CCAAACTAGAAATGCCATCTTC-3′) corresponds to nucleotides 7578 to 7599 of the normal human dystrophin cDNA (GenBank accession No. M18533, M17154, M18026) and 7569 to 7590 of mouse dystrophin cDNA (GenBank accession No. M68859). The 3′ primer (5′TCTGAATTCTTTCAATTCGAT-3′) corresponds to nucleotides 7863 to 7883 in the normal human dystrophin cDNA and 7854 to 7874 in the mouse dystrophin cDNA. To distinguish the mouse and human RT-PCR products, an EcoRV digestion was performed yielding two fragments of 145 and 160 bp for human mini-dystrophin and 305 bp for mouse dystrophin. 200 ng of DNAse-treated RNA was first reverse-transcribed by MoMLV RT and further amplified by Tth polymerase (33 cycles; Boehringer Mannheim).

PCR-mediated evaluation of adenovirus infection Mdx myoblasts were infected with the adenoviral vector (Ad-CMV-Dys) at different MOI ranging from 0.01 to 1000. Two days after the infection, DNA was extracted by a 1 h incubation at 37°C in a lysing solution (50 mm Tris-HCl pH 8.0, 20 mm EDTA, 1% SDS, 0.48 mg/ml proteinase K (Boehringer Mannheim)), followed by phenol– chloroform extraction and ethanol precipitation. To amplify the mini-dystrophin DNA, a 5′ primer (5′-GAGTACAGCACAGGAAACTG-3′) corresponding to the junction between exons 16 (nucleotide 2188) and 49 (nucleotide 7324) of normal dystrophin37 was designed. This junctional sequence is therefore only present in mini-dystrophin cDNA and mRNA. The 3′ primer (5′AGCAGGTACCTCCAACATCAAG-3′) corresponds to nucleotides 7600 to 7621 in the normal human dystrophin cDNA. 200 ng of DNA was amplified using Tth polymerase.

Acknowledgements This work was supported by the Association Franc¸aise contre les Myopathies (AFM), the Muscular Dystrophy Association (MDA), the Muscular Dystrophy Association of Canada (MDAC) and the Medical Research Council of Canada. Pierre-Alain Moisset is supported by a FRSQFCAR scholarship.

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6 Kochanek S et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independantly expressing both full-length dystrophin and ␤-galactosidase. Proc Natl Acad Sci USA 1996; 93: 5731–5736. 7 Engelhardt JF, Litzky L, Wilson JM. Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2A. Hum Gene Ther 1994; 5: 1217–1229. 8 Yang Y et al. Cellular immunity to viral antigens limits E1deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA 1994; 91: 4407–4411. 9 Yang Y et al. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet 1994; 7: 362–369. 10 Yang Y, Li Q, Ertl HCJ, Wilson JM. Cellular and humoral immune responses to viral antigens create barriers to lungdirected gene therapy with recombinant adenoviruses. J Virol 1995; 69: 2004–2015. 11 Vilquin JT et al. FK506 immunosuppression to control the immune reactions triggered by first generation adenovirusmediated gene transfer. Hum Gene Ther 1995; 6: 1391–1401. 12 Gue´rette B et al. Prevention of immune reactions triggered by first-generation adenoviral vectors by monoclonal antibodies and CTLA4Ig. Hum Gene Ther 1996; 7: 1455–1463. 13 Lochmu¨ller H et al. Transient immunosuppression by FK506 permits a sustained high level dystrophin expression after adenovirus mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic mice. Gene Therapy 1996; 3: 706–716. 14 Haecker SE et al. In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes. Hum Gene Ther 1996; 7: 1907–1914. 15 Chen HH et al. Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc Natl Acad Sci USA 1997; 94: 1645–1650. 16 Dedieu JF et al. Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses. J Virol 1997; 71: 4626–4637. 17 Kaplan JM et al. Humoral and cellular immune responses of nonhuman primates to long-term repeated lung exposure to Ad2/CFTR-2. Gene Therapy 1996; 3: 117–127. 18 Kohtz DS, Cole F, Wong M-L, Hsu M-T. Infection and inhibition of differentiation of human fetal skeletal myoblasts by adenovirus. Virology 1991; 184: 569–579. 19 Acsadi G et al. Cultured human myoblasts and myotubes show markedly different transducibility by replication defective adenovirus recombinant. Hum Gene Ther 1994; 1: 338–340. 20 Acsadi G et al. A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal mucle cells of different maturity. Hum Mol Gen 1994; 3: 579–584. 21 Feero WG et al. Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type I viral vectors. Hum Gene Ther 1997; 8: 371–380. 22 Karpati G et al. Myoblast transfer in Duchenne muscular dystrophy. Ann Neurol 1993; 34: 8–17. 23 Mendell JR et al. Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. New Engl J Med 1995; 333: 832–838.

24 Tremblay JP et al. Myoblast transplantation between monozygotic twin girl carriers of Duchenne muscular dystrophy. Neuromusc Dis 1993; 3: 583–592. 25 Partridge TA et al. Conversion of mdx myofibres from dystrophin negative to positive by injection of normal myoblasts. Nature 1989; 337: 176–179. 26 Vilquin JT et al. Successful myoblast allotransplantation in mdx mice using rapamycin. Transplantation 1995; 59: 422–449. 27 Kinoshita I et al. Myoblast transplantation in monkeys: control of immune response by FK506. J Neuropath Exp Neurol 1996; 55: 687–697. 28 Boulanger A, Asselin I, Tremblay JP. Role of non-major histocompatibility complex antigens in the rejection of transplanted myoblasts. Transplantation 1997; 63: 893–899. 29 McDiarmid SV et al. FK506 (tacrolimus) compared with cyclosporine for primary immunosuppression after pediatric liver transplantation. Transplantation 1995; 59: 530–536. 30 Kinoshita I et al. Transplantation of myoblasts from a transgenic mouse overexpressing dystrophin produced only a relatively small increase of dystrophin-positive membrane. Musc Nerve 1998; 21: 91–103. 31 Hallauer PL, Bradshaw HL, Hastings KEM. Complex fiber-typespecific expression of fast skeletal muscle troponin I gene constructs in transgenic mice. Development 1993; 119: 691–701. 32 Cossu G et al. In vitro differentation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells. Cell Differ 1980; 9: 357–365. 33 Wakeford S, Watt DJ, Partridge TA. X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD. Musc Nerve 1991; 14: 42–50. 34 Wirtz P, Loermans H, Rutten E. Effects of irradiation on regeneration in dystrophic mouse leg muscle. Br J Exp Pathol 1982; 63: 671–679. 35 Harris JB, Johnson MA, Karlsson E. Pathological responses of rat skeletal muscle to a single subcuteneous injection of a toxin from the venom of the Australian tiger snake, Notechis scutatus scutatus. Clin Exp Pharm Physiol 1975; 2: 383–404. 36 Huard J et al. Utilization of an antibody specific for human dystrophin to follow myoblast transplantation in nude mice. Cell Transplant 1993; 2: 113–118. 37 Acsadi G et al. Human dystrophin expression in mdx mice after injection of DNA constructs. Nature 1991; 352: 815–818.

References added in proof 38 Huard J, Acsadi G, Massie B, Karpati G. Gene transfer into skeletal muscles by isogenic myoblasts. Hum Gene Ther 1994; 5: 949–958. 39 Beauchamp JR, Pagel CN, Partridge TA. A dual-marker system for quantitative studies of myoblast transplantation in the mouse. Transplantation 1997; 63: 1794–1797. 40 Ito H, Hallauer PL, Hastings KEM, Tremblay JP. Prior culture with concanavalin A increases intramuscular migration of transplanted myoblast. Musc Nerve 1998; 21: 291–297. 41 Huard J et al. High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice. J Clin Invest 1994; 93: 586–599.