raav vector-mediated sarcogylcan gene transfer in a hamster model for limb girdle muscular dystrophy

Gene Therapy (1999) 6, 74 82 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt raav vector-mediated sarcogylcan gene transfer in a hamster model for limb girdle muscular dystrophy JLi 1, D Dressman 1, YP Tsao 1,6, A Sakamoto 2, EP Hoffman 1,3,4,5 and X Xiao 1 Departments of 1 Molecular Genetics and Biochemistry, 3 Human Genetics, 4 Pediatrics, and 5 Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 2 National Cardiovascular Center, Osaka, Japan; and 6 National Defense Medical Center, Taipei, Taiwan The limb girdle muscular dystrophies (LGMD) are a geneti- sarcoglycan-deficient hamster model (Bio14.6). We show cally and phenotypically heterogeneous group of degener- efficient delivery and widespread expression of -sarcoglyative neuromuscular diseases. A subset of the genetically can after a single intramuscular injection. Importantly, raav recessive forms of LGMD are caused by mutations in the vector containing the human -sarcoglycan cdna restored four muscle sarcoglycan genes (,, and ). The coding secondary biochemical deficiencies, with correct localizsequences of all known sarcoglycan genes are smaller ation of other sarcoglycan proteins to the muscle fiber than 2 kb, and thus can be readily packaged in recombi- membrane. Interestingly, restoration of -, as well as nant adeno-associated virus (raav) vectors. Previously, sarcoglycan was homogeneous and properly localized we have demonstrated highly efficient and sustained trans- throughout transduced muscle, and appeared unaffected duction in mature muscle tissue of immunocompetent ani- by dramatic overexpression of -sarcoglycan in the cytomals with raav vectors. In this report, we utilize recombi- plasm of some myofibers. These results support the feasinant AAV containing the -sarcoglycan gene for genetic bility of raav vector s application to treat LGMD by means complementation of muscle diseases using a - of direct in vivo gene transfer. Keywords: raav; sarcoglycan; limb girdle muscular dystrophy; Bio14.6 hamster Introduction The recombinant adeno-associated virus (raav) vector is a promising gene delivery system, which is derived from defective and nonpathogenic human parvoviruses. 1,2 The vector system offers a number of attractive featues for gene therapy, including transduction of both dividing and nondividing cells, the lack of CTL immune reactions and genomic integration. In contrast to other viral vectors, 3,4 raav is capable of efficiently bypassing the myofiber basal lamina and transducing mature muscle cells, which is particularly desirable for in vivo muscle gene transfer. We and others have successfully demonstrated that raav vectors harboring foreign genes, such as lacz, are capable of achieving highly efficient and sustained gene transfer in the mature muscle of immunocompetent animals for more than 1.5 years without detectable toxicity. 5 8 In addition, muscle tissue has been successfully used as a platform for raav-mediated therapeutic gene transfer for metabolic diseases and secreted proteins. 7 12 However, no experiments using raav vectors to restore the functional deficits in muscle tissue itself have been reported. The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of inherited neuromuscular diseases characterized by proximal muscular weakness and Correspondence: X Xiao, Department of Molecular Genetics and Biochemistry, Room W1213, BST, University of Pittsburgh, Pittsburgh, PA 15261, USA Received 28 July 1998; accepted 30 September 1998 variable progression of symptoms. The disease is caused by mutations in a number of responsible genes, 13 17 including sarcoglycan genes (LGMD 2D), (LGMD 2E), (LGMD 2C) and (LGMD 2F), respectively. 14,18 24 While the phenotype of the sarcoglycanopathies is variable, patients often show a severe progression, with fatality in the second or third decade. No effective treatment is available. 14,19 28 The sarcoglycans are relatively small (cdna 2 kb), transmembrane glycoproteins. The four commonly known sarcoglycans (,, and ) associate with each other in equal stoichiometry forming a hetero-tetramer, namely the sarcoglycan complex. Immunofluorescent studies of muscle samples from some LGMD patients and dystrophic animals have revealed that a primary deficiency of one component in the sarcoglycan complex leads to partial or complete absence of all the other sarcoglycan proteins on the sarcolemma. This suggests that these proteins are mutually dependent on each other for assembly and stability in the plasma membrane of muscle cells. 20,29 33 The sarcoglycan genes are expressed either exclusively (, and ) or predominantly ( ) in striated muscle. Recently a fifth sarcoglycan ( ) has been identified, however, with a broad gene expression pattern in various tissues including muscle. 34,35 Until recently, the development of gene therapy strategies to treat LGMD have been hampered by the lack of a proper animal model. The newly discovered mutation in the -sarcoglycan gene in a dystrophic hamster has provided a model for LGMD. 30,31 The inbred Syrian hamster Bio14.6 was identified in 1962, and shown to have

an autosomal recessive cardiomyopathy. 36 In addition to the pathology of cardiac muscle, the skeletal muscle of Bio14.6 hamsters also displays classical signs of myopathy, such as necrosis, central nucleation and variable size of myofibers. Recently, the genetic cause for the dystrophic disease in the hamster has been identified as a deletion in the promoter and the 5 untranslated region of the -sarcoglycan gene. 30,31 The deletion leads to the loss of mrna and consequently the absence of -sarcoglycan protein on the sarcolemma. 30,31 Consistent with the human sarcoglycanopathies, primary loss of -sarcoglycan also results in deficiency in all four sarcoglycans on the sarcolemma. The similarity of muscle histology, pathology and protein biochemistry between Bio14.6 hamster and human patients renders this animal an excellent model system to explore the possibility of gene therapy strategies for LGMD diseases. In the present study, we utilize the Bio14.6 hamster as the LGMD animal model and raav as the gene delivery vector to test the hypothesis that a genetic defect in muscle tissue can be complemented by means of direct intramuscular gene transfer. We report the first successful intramuscular injection of an raav vector containing the human -sarcoglycan cdna into the deficient Bio14.6 hamsters, and efficient transgene expression in muscle tissue from the vector. In addition, the human -sarcoglycan protein product has been correctly targeted to the muscle cell membrane, leading to the restoration of the sarcoglycan complex to the sarcolemma. Results Efficient transduction of regenerating muscle cells by raav vector Previously, we have demonstrated efficient and longterm transduction by AAV vectors in post-mitotic myofibers in mature muscle tissue of immunocompetent animals. 5,10 Snyder et al 37 have extended the in vivo studies from post-mitotic muscle tissue 5 to acutely regenerating muscle tissue, which was created by administration of a myotoxin, barium chloride. No experiment has been reported to date using raav vectors in pathological muscle caused by inherited defects. To test if raav vector efficiently transduces regenerating muscle in muscular dystrophies, we injected 30 l of raav-lacz vector (2 10 11 viral particles) into the hindleg gastrocnemius muscle of 40-day-old Bio14.6 hamsters. This muscle suffers extensive degeneration and regeneration caused by sarcoglycan deficiency. Hematoxylin and eosin (H&E) staining revealed profound regeneration with the characteristic centrally localized nuclei (Figure 1b), indicating muscle pathology in the Bio14.6 hamsters. Our in vivo vector injection results showed efficient transduction of regenerating muscle in these pathologic animals (Figure 1c and d), as visualized by X-gal staining, and H&E counterstaining of the muscle sections at 3 weeks after vector injection. As expected, the healthy control hamster F1B muscle was also efficiently transduced (Figure 1a). H&E staining in both dystrophic and normal muscle injected with the raav-lacz vector did not reveal cytotoxicity and lymphocyte infiltration, supporting the notion that raav vector carrying a foreign gene does not cause host CTL reactions. 5,7,38 Expression of -sarcoglycan gene in dystrophic hamster muscle from raav vector Encouraged by the results of efficient transduction in regenerating muscle with the AAV-LacZ reporter vector, we constructed an raav vector (paav-cmv- SG) containing the human -sarcoglycan gene under the control of CMV promoter (for details see Materials and methods). This promoter has been shown to exhibit elevated and sustained activity in muscle tissue in the context of raav. 5 7,39 42 Using this construct, we produced and purified high-titer, Ad-free AAV-CMV- SG viral vector stocks by the three-plasmid cotransfection method we have described previously. 42 To demonstrate the capability of producing -sarcoglycan protein by AAV-CMV- SG vector in infected cells, we initially performed an in vitro assay. Since no -sarcoglycan-defective muscle cell line was available, we tested the AAV-CMV- SG viral stock in human 293 cells, which do not express endogenous -sarcoglycan gene. We examined -sarcoglycan protein synthesis by Western blot using a polyclonal antibody, which recognizes a conserved region of -sarcoglycan in human, mouse and hamster. 31 Mock-infected 293 cells were used as a negative control (Figure 2a, lane 1), while normal mouse muscle tissue was used as a positive control (Figure 2a, lane 3). Human 293 cells infected with AAV-CMV- SG vector produced large quantities of -sarcoglycan at the expected molecular weight (Figure 2a, lane 2) compared with the normal mouse muscle tissue (Figure 2a, lane 3). This suggested that the expression cassette under the control of CMV promoter in 293 cells was highly active. The AAV-CMV- SG vector gene expression was then tested for its ability to complement sarcoglycan deficiency in muscle in vivo by intramuscular injection into the -sarcoglycan-defective Bio14.6 hamsters. Thirty microliters of AAV-CMV- SG vector (5 10 10 viral particles) was injected into the hindleg muscle (gastrocnemius) of 40-day-old Bio14.6 hamsters. The gastrocnemius muscle tissues were harvested at 3 weeks after vector injection from two hamsters and subjected to Western blot analysis. As shown in Figure 2b, muscle tissues from the two Bio14.6 hamsters injected with the AAV vector produced significant quantities of -sarcoglycan protein (Figure 2b, lanes 1 and 2). As expected, the dystrophic muscle untreated with AAV vector revealed no immunoblot signal for -sarcoglycan (Figure 2b, lane 3), while the control F1B hamster muscle showed a positive immunoblot signal (Figure 2b, lane 4). The -sarcoglycan protein levels from the vector-treated dystrophic muscle were approximately one-third of those of wildtype F1B hamster muscle. For Western blot, we solubilized a large piece of gastrocnemius muscle tissue complex from each animal. Thus, the 30% levels relative to normal muscle reflects localized regions of delivery by intramuscular injection and localized overexpression, and do not reflect the vector gene expression levels in individually transduced myofibers. To determine the extent of raav vector-mediated sarcoglycan expression in individual myofibers, immunofluorescent localization of -sarcoglycan was carried out on cryosections of muscle. The -sarcoglycan protein produced in muscle fibers following intramuscular injection of AAV-CMV- SG vector was correctly localized to sarcolemma in large groups of myofibers (Figure 3a). However, many myofibers showed overexpression of -sarco- 75

76 Figure 1 AAV-LacZ transduction in hindleg muscle of normal and Bio14.6 dystrophic hamsters. AAV-LacZ vector (30 l) was injected into the gastrocnemius muscle of hindlegs of 40-day-old animals. Three weeks after vector injection, the animals were killed and muscle sample processed with X-gal and H&E staining. (a) normal F1B hamster transduced with raav-lacz; (b) Bio14.6 hamster muscle, H&E staining. Note the numerous centrally localized nuclei indicative of muscle regeneration. (c) Bio14.6 hamster injected with AAV-LacZ, X-gal and H&E staining. Note that X-gal staining was too prominent to see the centrally localized myofiber nuclei after H&E staining. (d) An enlarged area of (c). Note the numerous LacZ-positive centrally nucleated myofibers. Also not the heterogeneous sizes of the myofibers. Original magnification: (a) and (c) at 4 objective and (b) and (d) at 10 objective. glycan protein, leading to substantial staining in the cytoplasm of these fibers (see leftward arrow in Figure 3a). Overexpression of the human -sarcoglycan gene did not result in any discernable histopathology in these myofibers. Restoration of the sarcoglycan complex To assess the ability of the -sarcoglycan protein to restore the membrane localization of the other components in the sarcoglycan complex, immunofluorescent staining was used to examine further the vector-treated muscle. If the -sarcoglycan gene delivered by the raav vector is functional, the gene product should correctly localize itself to the sarcolemma and form a stable sarcoglycan complex with other sarcoglycan proteins (, and ). Flash-frozen, unfixed muscle samples from Bio14.6 hamsters were cryosectioned at 3 weeks after intramuscular vector injection. To detect vector-mediated -sarcoglycan gene expression, the fresh muscle frozen sections (5 m) were immediately blocked in 10% horse serum/pbs. Monoclonal antibody against human -sarcoglycan was used in conjugation with Cy-3-labeled anti-mouse antibody for detection. Immunostaining of adjacent cryosections for -sarcoglycan (Figure 3a) and -sarcoglycan (Figure 3b) showed that vector-mediated exogenous -sarcoglycan gene expression led to restoration of endogenous -sarcoglycan in all myofibers. This analysis showed a number of important points. First, myofibers expressing relatively low levels of -sarcoglycan still achieved levels of -sarcoglycan, which are indistinguishable from normal muscle (Figures 3 and 4). Second, overexpression of -sarcoglycan in the cytoplasm of some myofibers did not lead to incorrect location of -sarcoglycan (see rightward arrow, Figure 3a and b). Thus, the secondary deficiency of -sarcoglycan 20,29 33 was rescued by raav vector delivery, independent of the levels of -sarcoglycan expression. Adjacent cryosections from normal hamster, dystrophic hamster and dystrophic hamster treated with AAV-CMV- SG vector were immunostained for -, and -sarcoglycan (Figure 4). Secondary deficiency of sarcoglycan was also completely rescued by the vector delivery. Similar to the results of -sarcoglycan, overexpression of -sarcoglycan did not disrupt the normal membrane localization of -sarcoglycan (Figure 4). In

Figure 2 Western analysis of -sarcoglycan gene expression in vitro and in vivo. (a) -Sarcoglycan gene expression in human 293 cells infected in vitro with AAV-CMV- SG vector. Normal mouse muscle tissue was used as a positive control. Note the robust gene expression of the raav vector in 293 cells. (b) -Sarcoglycan gene expression in vivo in Bio14.6 hamster gastrocneumius muscle after AAV-CMV- SG vector injection. Normal hamster muscle was used as a positive control. addition, the overexpression of human -sarcoglycan evidently did not appear to cause cytotoxicity and lymphocyte infiltration, consistent with the previous findings that there is a lack of host CTL response to raav-transduced cells. 5 8 Discussion We have shown the successful use of raav as a gene vector for genetic and biochemical rescue of the Bio14.6 hamster, a homologous animal model for LGMD. We have shown efficient gene transfer and expression of the human -sarcoglycan gene in the -sarcoglycan-deficient hamster muscle. Expression of -sarcoglycan from the raav vector led to the restoration of the secondary biochemical defects; specifically, both - and -sarcoglycan were localized to the myofiber membrane in a manner indistinguishable from normal hamster muscle. Although we did not perform immunostaining of the fourth sarcoglycan component ( ) due to the unavailability of a working antibody for this hamster protein, the biochemical property of the sarcoglycan complex suggests that the sarcoglycan must have also been correctly reconstituted into the complex, because the sarcoglycan complex cannot be stably formed without the presence of all four proteins. In fact, successful adenoviral vector-directed human -sarcoglycan gene transfer in Bio14.6 hamster has been recently reported. 43 These experiments indicate that exogenously delivered human -sarcoglycan gene is biochemically functional in the dystrophic hamster muscle cells. Further studies are needed to test for physiological functional improvement of the treated muscle, such as increase of muscle strength and protection of muscle cell membrane integrity, afer raav-mediated -sarcoglycan gene delivery. However, our results prove the concept that a deficiency of muscle function caused by a recessive genetic mutation can be complemented by intramuscular injection of an raav vector carrying the corresponding gene. raav is currently the best available candidate vector for gene therapy of inherited muscle disease. The vector system offers numerous advantages for in vivo muscle gene transfer. However the 5 kb packaging limit precludes its utility for some of the more common muscle diseases. For example, the most common muscular disease is Duchenne muscular dystrophy caused by dystrophin deficiency. 44 The cdna for dystrophin protein is larger than 10 kb. 45 However, all the sarcoglycan genes are small (cdnas 2 kb) and can be readily accommodated into raav vector, as we have shown in the present study. The high efficiency of transduction that we have shown here is probably a consequence of the small particle sizes of AAV (20 nm), which can readily bypass the surrounding basal laminar barrier and infect mature myofibers. Small viral particle size also offers the advantage of bypassing the capillary blood vessel pores when delivered systemically. 41 In contrast, adenoviral and herpes viral vectors have much larger particle size and have encountered difficulties in bypassing the basal laminar barrier to transduce mature muscle cells. 3,4 Besides mature muscle, raav has been shown to transduce efficiently regenerating muscle cells. In this study, we observed no significant difference in terms of transduction efficiency between healthy mature muscle and regenerating muscle in both normal and Bio14.6 hamsters. Consistently, experiments in normal or dystrophic mdx mouse muscle revealed identical transduction efficiency by raav-lacz vector, as quantified by -galactosidase activities (J Huard, personal communications). The ability to transduce efficiently both mature and regenerating muscle cells is important for the success of gene therapy in muscular dystrophies, owing to the fact that the majority of the dystrophic patients are diagnosed at the age when most muscle cells have already matured and manifested variable extents of regeneration. Intramuscular injection of an raav vector containing human -sarcoglycan gene into the defective hamster muscle resulted in efficient gene transfer and expression. The human -sarcoglycan protein was correctly targeted to the muscle cell membrane. Moreover, we showed that 77

78 Figure 3 Widespread expression of -sarcoglycan and restoration of -sarcoglycan after intramuscular injection of AAV-CMA- SG vector. (a) -Sarcoglycan antibody staining of AAV-CMV- SG vector injected Bio14.6 dystrophic muscle. (b) -Sarcoglycan antibody staining of an adjacent cryosection showing the same microscopic field as in (a). The -sarcoglycan protein shows variable expression, with some fibers showing overexpression and cytoplasmic storage of the protein (a, leftward arrow) and some fibers showing normal level of the protein (a, rightward arrow). However, the restoration of secondary -sarcoglycan deficiency was seen to be homogeneous, independent of overexpressed levels of -sarcoglycan (b, leftward arrow). (c) A different area of -sarcoglycan antibody staining of AAV-CMV- SG vector injected Bio14.6 dystrophic muscle. In the upper right corner, some positive myofibers (arrow) are surrounded by the negative ones (arrowhead). The cell membrane staining is apparent.

79 Figure 4 Restoration of the entire sarcoglycan complex to wild-type levels after -sarcoglycan gene delivery by AAV-CMV- SG vector. Muscle cryosections from normal, dystrophic and AAV-CMV- SG-treated dystrophic hamsters were immunofluorescently stained for -, - and -sarcoglycan ( -SG, -SG and -SG), respectively. Note that the -sarcoglycan monoclonal antibody is human-specific, and barely detects hamster -sarcoglycan. The Bio14.6 dystrophic hamster shows both primary -sarcoglycan deficiency and secondary deficiency of - and -sarcoglycan. raav vector-mediated intramuscular gene delivery results in high-level expression of human -sarcoglycan gene in large regions of the muscle, and leads to the restoration of wild-type levels of - and -sarcoglycan in the sarcoglycan complex to the myofiber membrane. the secondary biochemical deficiencies of - and -sarcoglycan were corrected. Importantly, we found that overexpression of -sarcoglycan by the strong CMV promoter led to apparent saturation of membrane binding sites and cytoplasmic staining for -sarcoglycan in some myofibers. However, this ectopic localization of -sarcoglycan had no apparent effect on the localization of - and sarcoglycan. Immunostaining of these two proteins showed uniform membrane distribution regardless of the variable levels of -sarcoglycan, indicating that oversupply of one component of the sarcoglycan complex, at least in the case of -sarcoglycan, does not interfere with the formation of the entire complex. In this situation, the component shifted from a rate-limiting factor in the untreated dystrophic muscle to a non-rate-limiting factor in the vector-treated muscle. Since the four components are in equal molar ratio in the complex, a physiologically rationed supply of a single component should potentially keep the formation of the entire sarcoglycan complex at the normal level. A second important finding is that overexpression of the human -sarcoglycan protein in the deficient hamster muscle did not cause cytotoxicity of the muscle cells, or detectable immune responses during the 3 week period examined. Lymphocyte infiltration associated with the CTL reactions in normally observed starting in the first 2 weeks of vector injection in the case of other vectors in adult immunocompetent animals. Although further long-term observation is warranted, it is unlikely that CTL reaction will be initiated after this period, especially given the fact that raav vector carrying completely foreign genes, such as bacterial lacz and jellyfish GFP genes, does not illicit any CTL reaction to the transduced cells regardless of the duration of the studies. 5 Based on previous in vivo studies, we anticipate that raav-mediated -sarcoglycan gene transfer in the Bio14.6 hamster muscle will display sustained expression due to vector integration into the host chromosomes. 5,7,46 Even though overexpression of -sarcoglycan gene did not cause detectable adverse effects during the period tested in this study, regulated muscle-specific expression of the transgene is obviously a highly preferable feature for human gene therapy in the future. Recently, we constructed an raav vector containing the human -sarcoglycan gene under the control of a muscle-specific creatine kinase (MCK) promoter. 47 In vivo examination of this construct is underway in the Bio14.6 hamster. The success of gene therapy for LGMD in this relevant animal

80 model will bring us one step closer to the genetic treatment of the human disease by means of direct intramuscular injection of the raav vectors. However, future applications directed towards treatment of human patients must address the large target organ (muscle is approximately 30% of body mass) and cell-autonomous nature of the sarcoglycan defect. Systemic delivery and muscle-specific expression should overcome this barrier. Materials and methods Production of raav vectors An raav vector containing human -sarcoglycan cdna under the control of the CMV promoter was constructed as follows. First -sarcoglycan cdna was amplified by RT-PCR from human muscle biopsy cdna, using primers -SG-For (5 TCCTTCAGAGCTGCTGCTCAGCA CGCCC 3 ) and -SG-Rev (5 CCCGTTTGTTCATT GCCCATCAGGCCC 3 ). The amplified cdna was then cloned into their pgem-t Easy Vector (Promega, Madison, WI, USA) and subsequently excised by NotI digestion. The gel purified NotI fragment was subcloned into the NotI sites of an AAV vector plasmid pxx-uf1, which is a derivative of an raav vector plasmid ptr-uf1 48 with the substitution of the original plasmid backbone pbluescript by pembo. The resulting vector plasmid was named paav-cmv- SG. AAV-CMV- SG viral vector stocks were produced by cotransfection methods as previously described. 42 Briefly, 1 2 h before transfection, each 15-cm plate of human 293 cells, at 70 80% confluence, was fed with 25 ml fresh IMDM medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) without antibiotics. A total of 50 g plasmid DNA (25 g of XX6, 6 g of XX2 and 19 g of AAV-CMV- SG) was dissolved in 2 ml of 0.25 m CaCl 2 and then quickly mixed with 2 ml of HBS buffer (50 mm Hepes, 280 mm NaCl and 1.5 mm Na 2 HPO 4, ph 7.12) and added to the cells. At 8 12 h after transfection, the medium was replaced with fresh DMEM medium (Gibco) containing 10% FBS and antibiotics. The transfected cells containing raav viral particles were harvested at 48 h after transfection. The raav viral vector was purified twice through CsCl density gradient purification according to the perviously published methods. 49 The vector titers of viral particle number were determined by the DNA dot-blot method, 49 and were in the range of 2 5 10 12 viral particles per milliliter. In vivo vector delivery into muscle tissue The dystrophic hamster Bio14.6 and the normal control hamster F1B were purchased from Bio Breeders (Fitchburg, MA, USA) and handled in accordance with the institutional guidelines of the University of Pittsburgh. Before virus injection, 40-day-old hamsters were anesthetized with 2.5% Avertin (Aldrich, Milwaukee, WI, USA) intraperitoneally. Either 30 l of AAV-Lacz (2 10 11 viral particles) or 30 l of AAV-CMV- SG (5 10 10 viral particles) was injected into the hind leg gastrocnemius muscles percutaneously. At 3 weeks after intramuscular vector injection, the hamsters were killed and the muscle tissues were harvested and rapidly frozen in isopentane cooled in liquid nitrogen for further analysis. Western analysis Western analysis was carried out according to previously published methods. 50 Briefly, 293 cells injected with AAV-CMV- SG were lysed in RIPA buffer (10 mm Tris- Cl, ph 8.2, 1% Triton X-100, 1% SDS and 150 mm NaCl). For hamster muscle samples, 100 mg gastrocnemius muscle was homogenized and lysed in the RIPA buffer. The samples were separated on 10% SDS-PAGE and transferred on to nitrocellulose membrane. After blocking in 10% nonfat dry milk in TBS buffer (50 mm Tris-Cl, ph 7.5, 200 mm NaCl) for 1 h, the membranes were incubated with primary antibodies in TBS containing 0.5% Tween- 20 at room temperature for 1 h. The polyclonal antibody recognizing a conserved epitope of human, mouse and hamster -sarcoglycan was used in this experiment with a 1:5000 dilution. 31 Following primary antibody incubation and rinses, the membranes were incubated with the secondary antibody, a goat anti-rabbit antibody conjugated with horseradish peroxidase (Sigma, St Louis, MD, USA) with 1:5000 dilution in 2% dry milk and TBS buffer. After 1 h of antibody incubation and three washes with TBS buffer containing 0.5% Tween-20 and once with TBS, the -sarcoglycan protein band was visualized with chemiluminescence reagent (DuPont, Boston, MA, USA) and exposed to radiographic film. Immunofluorescent and X-gal staining Cryostat sectioning of the muscle tissue was performed at 5 m thickness with an IECMinotone (International Equipment Company, Needham Heights, MA, USA). The sections were then fixed for X-gal staining according to a published method. 5 For immunofluorescent staining, 22 the unfixed muscle cryosections were immediately blocked in 10% horse serum and PBS at room temperature for 1 h without fixation. Monoclonal antibodies against - and -sarcoglycans (Novocatra Laboratories, Newcastle, UK), or polyclonal antibody to -sarcoglycan 29 were diluted 1:100 in 10% horse serum/pbs, and incubated with cryosections 3 h at room temperature. After washes, the sections were incubated with Cy-3-labeled anti-mouse or anti-rabbit secondary antibodies at 1:500 dilution in 10% horse serum/pbs (Jackson Immunoresearch Laboratories). After washes, the samples were mounted in 90% glycerol/pbs or Gelmount (Fisher, Pittsburgh, PA, USA). Photographs were taken with a Nikon Microphot-FXA microscope, using an Optronics DE1750 color integrating digital camera. Acknowledgements We thank Dr Eijiro Ozawa for the polyclonal antibody to -sarcoglycan and Dr T Toyo-Oka for the polyclonal antibody to -sarcoglycan. We thank Dr Johny Huard and Mr Ryan Pruchnic for assistance with photography. This work is supported by the University of Pittsburgh (X Xiao), and Muscular Dystrophy Association USA (EP Hoffman). EP Hoffman is an Established Investigator of the American Heart Association. References 1 Berns KI, Linden RM. The cryptic life style of adeno-associated virus. Bioessays 1995; 17: 237 245. 2 Berns KI. Parvoviridae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM (eds). Virology. Lippincott- Raven: Philadelphia, 1996, pp 2173 2198.

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