Analysis of Muscle Creatine Kinase Regulatory Elements in Recombinant Adenoviral Vectors
Authors: Michael A. Hauser, Ann Robinson, Dennis Hartigan-O'Connor, DeeAnn Williams-Gregory, Jean N. Buskin, Steve Apone, Christopher J. Kirk, Stephen Hardy, Stephen D. Hauschka, Jeffrey S. Chamberlain
Journal: Molecular Therapy (2000)

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection. Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection. IntroductionNumerous challenges remain for the development of effective gene therapy protocols for muscle, most notably the difficulty of achieving efficient gene delivery to this large and diffuse tissue. Direct intramuscular injection of various vectors including adenovirus (1Stratford-Perricaudet L.D. Makeh I. Perricaudet M. Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart.J. Clin. Invest. 1992; 90: 626-630Crossref PubMed Scopus (573) Google Scholar) and adeno-associated virus (AAV) (2Xiao X. Li J. Samulski R.J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.J. Virol. 1996; 70: 8098-8108Crossref PubMed Google Scholar, 3Xiao X. Li J. Samulski R.J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J. Virol. 1998; 72: 2224-2232Crossref PubMed Google Scholar) results in efficient transduction of cells near the site of injection; however, the limited diffusion of these vectors within the target tissue would require a very large number of injections to treat large muscles. Promising methods for systemic administration of viral vectors are being developed (4Welling T.H. et al.Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb.Hum. Gene Ther. 1996; 7: 1795-1802Crossref PubMed Scopus (16) Google Scholar, 5Drazan K.E. Hebebrand D. Shaked A. Jones N.F. Gene transfer into nerve and muscle by isolated limb perfusion or during replantation.J. Reconst. 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Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.J. Virol. 1998; 72: 4212-4223Crossref PubMed Google Scholar). Development of safe and efficient gene transfer to muscle will undoubtedly require the use of improved vector backbones, such as "gutted" adenoviruses (8Kumar-Singh R. Chamberlain J.S. Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells.Hum. Mol. Genet. 1996; 5: 913-921Crossref PubMed Scopus (157) Google Scholar, 9Clemens P.R. et al.In vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes.Gene Ther. 1996; 3: 965-972PubMed Google Scholar, 10Hartigan-O'Connor D. Chamberlain J.S. Progress toward gene therapy of Duchenne muscular dystrophy.Semin. Neurol. 1999; 19: 323-332Crossref PubMed Scopus (14) Google Scholar), or alternate vector systems, such as AAV (2Xiao X. Li J. Samulski R.J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.J. Virol. 1996; 70: 8098-8108Crossref PubMed Google Scholar). The safety and effectiveness of systemic vector delivery could also be improved by the development of methods to ensure tissue-specific transgene expression. One approach is to create a vector expressing modified coat proteins that preferentially direct uptake by muscle cells and/or exclude uptake by liver cells. Such modified-tropism adenoviruses are under development, but have not as yet achieved the required degree of tissue specificity (11Krasnykh V.N. Mikheeva G.V. Douglas J.T. Curiel D.T. Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.J. Virol. 1996; 70: 6839-6846Crossref PubMed Google Scholar, 12Douglas J.T. Curiel D.T. Strategies to accomplish targeted gene delivery to muscle cells employing tropism-modified adenoviral vectors.Neuromusc. 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In terferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter.Hum. Gene Ther. 1995; 6: 1291-1297Crossref PubMed Scopus (144) Google Scholar, 16Loser P. Jennings G.S. Strauss M. Sandig V. Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: Involvement of NFkappaB.J. Virol. 1998; 72: 180-190Crossref PubMed Google Scholar).A number of first-generation adenoviruses containing tissue-specific transcriptional regulatory elements have been reported. A recombinant adenovirus containing hepatitis B virus regulatory elements directs strong, liver-specific transgene expression (17Sandig V. Loser P. Lieber A. Kay M.A. Strauss M. HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene.Gene Ther. 1996; 3: 1002-1009PubMed Google Scholar); myocardial-specific expression has been reported for adenoviral vectors containing ventricle-specific myosin light chain-2 and alpha-myosin heavy chain promoters (18Franz W.M. Rothmann T. Frey N. Katus H.A. Analysis of tissue-specific gene delivery by recombinant adenoviruses containing cardiac-specific promoters.Cardiovasc. Res. 1997; 35: 560-566Crossref PubMed Scopus (80) Google Scholar); and an adenovirus containing the pancreatic amylase promoter exhibited pancreas-specific expression (19Dematteo R.P. et al.Engineering tissue-specific expression of a recombinant adenovirus: Selective transgene transcription in the pancreas using the amylase promoter.J. Surg. Res. 1997; 72: 155-161Abstract Full Text PDF PubMed Scopus (28) Google Scholar). In addition, tumor-specific expression from adenoviral vectors has been proposed as a safe and efficient way to target tumor cells for destruction. To this end, tumor-specific adenoviruses have been reported using the carcinoembryonic antigen promoter (active in gastric tumors) (20Tanaka T. et al.Adenovirus-mediated gene therapy of gastric carcinoma using cancer-specific gene expression in vivo.Biochem. Biophys. Res. 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Commun. 1998; 244: 455-462Crossref PubMed Scopus (94) Google Scholar), and the prostate-specific antigen promoter (active in prostate tumor cells) (25Gotoh A. et al.Development of prostate-specific antigen promoter-based gene therapy for androgen-independent human prostate cancer.J. Urol. 1998; 160: 220-229Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).The muscle creatine kinase (MCK) gene is highly active in all striated muscles. MCK is the most abundant nonmitochondrial mRNA that is expressed in all skeletal muscle fiber types (26Welle S. Bhatt K. Thornton C.A. In ventory of high-abundance mRNAs in skeletal muscle of normal men.Genome Res. 1999; 9: 506-513PubMed Google Scholar) and is also highly active in cardiac muscle (27Amacher S.L. Buskin J.N. Hauschka S.D. Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.Mol. Cell. 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MCK gene regulatory regions display striated muscle-specific activity and have been extensively characterized in vitro and in vivo (27Amacher S.L. Buskin J.N. Hauschka S.D. Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.Mol. Cell. Biol. 1993; 13: 2753-2764Crossref PubMed Scopus (135) Google Scholar, 30Jaynes J.B. Chamberlain J.S. Buskin J.N. Johnson J.E. Hauschka S.D. Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts.Mol. Cell. Biol. 1986; 6: 2855-2864Crossref PubMed Scopus (116) Google Scholar, 31Trask R.V. Strauss A.W. Billadello J.J. Developmental regulation and tissue-specific expression of the human muscle creatine kinase gene.J. Biol. Chem. 1988; 263: 17142-17149PubMed Google Scholar, 32Cserjesi P. Lilly B. Bryson L. Wang Y. Sassoon D.A. Olson E.N. MHox: A mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer.Development. 1992; 115: 1087-1101PubMed Google Scholar, 33Shield M.A. Haugen H.S. Clegg C.H. Hauschka S.D. E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice.Mol. Cell. Biol. 1996; 16: 5058-5068Crossref PubMed Scopus (99) Google Scholar). The major known regulatory regions in the MCK gene include a muscle-specific enhancer located approximately 1.1 kb 5' of the transcription start site in mouse and a 358-bp proximal promoter (33Shield M.A. Haugen H.S. Clegg C.H. Hauschka S.D. E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice.Mol. Cell. Biol. 1996; 16: 5058-5068Crossref PubMed Scopus (99) Google Scholar, 34Jaynes J.B. Johnson J.E. Buskin J.N. Gartside C.L. Hauschka S.D. The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer.Mol. Cell. Biol. 1988; 8: 62-70Crossref PubMed Scopus (170) Google Scholar). Additional sequences that modulate MCK expression are distributed over a 3.3-kb region 5' of the transcriptional start site and in the 3.2-kb first intron (35Sternberg E.A. Spizz G. Perry W.M. Vizard D. Weil T. Olson E.N. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene.Mol. Cell. Biol. 1988; 8: 2896-2909Crossref PubMed Scopus (174) Google Scholar, 36Johnson J.E. Wold B.J. Hauschka S.D. Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.Mol. Cell. Biol. 1989; 9: 3393-3399Crossref PubMed Scopus (160) Google Scholar), and constructs containing these 3.3- and 6.5-kb regions direct high-level gene expression in skeletal and cardiac muscle in transgenic mice (36Johnson J.E. Wold B.J. Hauschka S.D. Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.Mol. Cell. Biol. 1989; 9: 3393-3399Crossref PubMed Scopus (160) Google Scholar, 37Cox G.A. et al.Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.Nature. 1993; 364: 725-729Crossref PubMed Scopus (264) Google Scholar, 38Phelps S.F. et al.Expression of full-length and truncated dystrophin minigenes in transgenic mdx mice.Hum. Mol. Genet. 1995; 4: 1251-1258Crossref PubMed Scopus (264) Google Scholar, 39Rafael J.A. Cox G.A. Corrado K. Jung D. Campbell K.P. Chamberlain J.S. Forced expression of dystrophin deletion constructs reveals structure-function correlations.J. Cell Biol. 1996; 134: 93-102Crossref PubMed Scopus (157) Google Scholar). While recombinant MCK promoter constructs of considerably less than 3 kb exhibit high activity after transient transfection into myogenic cell lines (27Amacher S.L. Buskin J.N. Hauschka S.D. Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.Mol. Cell. Biol. 1993; 13: 2753-2764Crossref PubMed Scopus (135) Google Scholar), some of these same constructs display greatly reduced activity relative to the 3.3-kb promoter when tested in transgenic mice (28Donoviel D.B. Shield M.A. Buskin J.N. Haugen H.S. Clegg C.H. Hauschka S.D. Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.Mol. Cell. Biol. 1996; 16: 1649-1658Crossref PubMed Scopus (86) Google Scholar, 33Shield M.A. Haugen H.S. Clegg C.H. Hauschka S.D. E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice.Mol. Cell. Biol. 1996; 16: 5058-5068Crossref PubMed Scopus (99) Google Scholar). Of particular interest for potential gene therapy purposes is an ~570-bp construct containing the enhancer ligated to the proximal promoter region; these two regions exhibit strong synergistic interactions in vivo while little synergism is observed in vitro (33Shield M.A. Haugen H.S. Clegg C.H. Hauschka S.D. E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice.Mol. Cell. Biol. 1996; 16: 5058-5068Crossref PubMed Scopus (99) Google Scholar). Mutagenic approaches have also been used to create more highly active MCK regulatory regions; this has been particularly successful within the MCK enhancer where the substitution of a more active E-box and mutagenesis of a region flanking one of the enhancer E-boxes have each created more active enhancers (Apone and Hauschka, unpublished).Recently, several groups have used portions of the MCK gene in attempts to generate muscle-specific adenoviral (Ad) vectors. An Ad vector containing the MCK upstream enhancer plus the human ventricular slow muscle myosin light chain 1 promoter expressed poorly in muscle, and the specificity and activity of this chimeric regulatory cassette were dramatically reduced by the presence of flanking adenoviral sequences (40Shi Q. Wang Y. Worton R. Modulation of the specificity and activity of a cellular promoter in an adenoviral vector.Hum. Gene Ther. 1997; 8: 403-410Crossref PubMed Scopus (59) Google Scholar). In contrast, an adenovirally delivered 1.35-kb mouse genomic MCK promoter/enhancer fragment was not suppressed by Ad sequences (41Larochelle N. et al.Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.Gene Ther. 1997; 4: 465-472Crossref PubMed Scopus (43) Google Scholar). This latter virus directed expression of luciferase primarily in muscle tissue, although cell-type specificity was not examined and the strength of the 1.35-kb fragment was not compared with the larger 3.3- and 6.5-kb MCK regulatory regions. Such larger MCK regulatory fragments have previously been used by our group and others to direct muscle-specific dystrophin expression from "gutted" adenoviral vectors that have extremely large cloning capacities (8Kumar-Singh R. Chamberlain J.S. Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells.Hum. Mol. Genet. 1996; 5: 913-921Crossref PubMed Scopus (157) Google Scholar, 9Clemens P.R. et al.In vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes.Gene Ther. 1996; 3: 965-972PubMed Google Scholar). However, conventional Ad vectors and AAV often cannot accommodate these MCK gene fragments, which are typically used in combination with an intron, polyadenylation signal, and a transgene. In this report, we describe the development and testing of a series of small, highly active MCK regulatory cassettes. Several of these constructs contain a mutagenized MCK enhancer that significantly increases gene expression plus a truncated MCK promoter region. We show that these regulatory cassettes retain strict muscle-specific expression in adult mice when delivered by a first-generation adenoviral vector.Materials and MethodsConstruction of MCK expression cassettesAll MCK sequences were derived from a 3355-bp murine genomic fragment (30Jaynes J.B. Chamberlain J.S. Buskin J.N. Johnson J.E. Hauschka S.D. Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts.Mol. Cell. Biol. 1986; 6: 2855-2864Crossref PubMed Scopus (116) Google Scholar) extending from –3348 to +7 relative to the transcriptional start site. The complete nucleotide sequence of this genomic fragment has been submitted to GenBank (Accession No. AF188002). CK3 includes this 3355-bp sequence, including the wild-type enhancer extending from positions –1256 to –1051 relative to the transcriptional start site. CK2 includes the MCK regions –1256 to +7. The 2RS5 enhancer was constructed with standard methods of oligomer-mediated site-specific mutagenesis. This mutant enhancer was PCR amplified with primers that created a KpnI site upstream of nucleotide –1256 and an ApaI site downstream of nucleotide –1050. This product was ligated to the naturally occurring ApaI site located at –80 nucleotides to construct CK4. A promoter fragment extending from nucleotide –358 to 3' of the transcriptional start site was PCR amplified with a primer that incorporated an ApaI site upstream of nucleotide –358. This PCR product was digested with ApaI and ligated into the ApaI site of CK4 to obtain CK6. CK5 is similar to CK2 with MCK sequences from –1256 to +7, but it is smaller by 148 bp, omitting MCK sequences from –1091 to –944, and it contains the 2RS5 mutations. The CMV promoter used in the plasmid expression cassettes extends from nucleotides –525 to +1 relative to the transcriptional start site (42Boshart M. Weber F. Jahn G. Dorsch-Hasler K. Fleckenstein B. Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.Cell. 1985; 41: 521-530Abstract Full Text PDF PubMed Scopus (842) Google Scholar). All expression cassettes also contain the minx intron (43Niwa M. Rose S.D. Berget S.M. In vitro polyadenylation is stimulated by the presence of an upstream intron.Genes Dev. 1990; 4: 1552-1559Crossref PubMed Scopus (228) Google Scholar) and the SV40 late polyadenylation signal.VirusesThe recombinant MCK adenoviruses AdCK5lacZ and AdCK6lacZ were constructed using a cre–lox recombination method (44Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. Construction of adenovirus vectors through Cre-lox recombination.J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). Briefly, the expression cassettes from Fig. 2 were cloned between the SacII and ClaI sites of the shuttle vector pAdlox, and, along with the recombinant adenovirus Ψ8, the resulting plasmid was transfected into 293 cells producing Cre recombinase. After the desired construct was generated by cre-mediated recombination, clonal isolates were identified by plaque purification and Southern analysis. In recombinant viruses, transgenes replace E1 sequences from nucleotides 359–3328, and E3 sequences are deleted from nucleotides 28,133–30,818. AdCMVlacZ was constructed by inserting the CMV expression cassette in Fig. 2 into the BglII site of pAdBglII. This plasmid was then cotransfected into 293 cells with dl7001 and recombinants were plaque purified. The first-generation adenovirus H5.010CBlacZ contains lacZ under control of the chicken β-actin promoter and CMV enhancer (45Yang Y. Nunes F.A. Berencsi K. Furth E.E. Gonczol E. Wilson J.M. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.Proc. Natl. Acad. Sci. USA. 1994; 91: 4407-4411Crossref PubMed Scopus (1522) Google Scholar) and was obtained from the University of Michigan viral vector core laboratory. Viral stocks were prepared by standard methods: after amplification in 293 cells and purification on two sequential CsCl density gradients, virus was desalted by gel filtration or dialysis and stored at –20°C in phosphate-buffered saline containing 10% glycerol. Viral titers ranged from 4 × 109 to 2 × 1011 plaque-forming units (pfu)/ml, with particle/pfu ratios from 40 to 110.Tissue cultureMM14 myogenic cells were cultured as previously described (46Neville C. Rosenthal N. McGrew M. Bogdonova N. Hauschka S. Skeletal muscle cultures.In Methods in Cell Biology.Methods in Muscle Biology.Vol.52. Academic Press, San Diego1997: 85-116Google Scholar). Briefly, actively dividing cultures were fed every 12 h with Ham's F10 medium supplemented with 0.141 g/liter calcium chloride dihydrate, 15% horse serum, and 2 ng/ml human recombinant basic fibroblast growth factor (FGF). Cells at a density of 3–5 × 105 per 100-mm dish were transfected with 8 μg of the plasmid being tested along with 2 μg of a placental alkaline phosphatase reference plasmid, pSV2APAP (47Henthorn P. Zervos P. Raducha M. Harris H. Kadesch T. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression.Proc. Natl. Acad. Sci. USA. 1988; 85: 6342-6346Crossref PubMed Scopus (133) Google Scholar), by calcium phosphate precipitation (48Neville C. Rosenthal N. Hauschka S. DNA transfection of cultured muscle cells.In Methods in Cell Biology.Methods in Muscle Biology.Vol.52. Academic Press, San Diego1997: 405-422Google Scholar). Cells were then induced to differentiate by withdrawal of FGF and reduction of serum to 1.5% and 30 h later were lysed into Tris-buffered saline containing 0.5% Triton X-100. Extracts were assayed for β-galactosidase activity using the fluorescent substrate 4-methylumbelliferyl-β-D-galactoside in conjunction with the Hoeffer TKO fluorometer (49MacGregor G.M. Nolan G.P. Fiering S. Roederer M. Herzenberg L.A. Use of E. coli lacZ (?-galactosidase) as a reporter gene. In Methods in Molecular Biology.Gene Transfer and Expression Protocols.Vol.7. Human Press, Clifton, NJ1991: 217-225Google Scholar). Extracts were also assayed for placental alkaline phosphatase in order to normalize for transfection efficiency.MiceSix-week-old C57Bl/10mdx mice (dystrophin-negative strain) obtained from a breeding colony at the University of Michigan were anesthetized by intraperitoneal injection of avertin (200 mg/kg tribro-moethanol, 200 mg/kg tert-amyl alcohol in PBS), and hair was removed from the surgical site with clippers. For intramuscular (im) viral injections, the tibialis anterior muscle was injected transdermally with 1 × 109 pfu in a volume of 25 μl. For im plasmid DNA injections, an incision was made in the skin to expose the rectus femoralis muscle, 50 μg endotox-in-free plasmid was injected in a volume of 25 μl PBS, and the skin was closed with wound clips. All im injections were carried out with a 30-gauge needle fitted with plastic tubing to ensure a 2-mm depth of injection. For intravenous injections, 3-week-old mdx mice were injected with 2 × 109 pfu in the tail vein with a 30-gauge needle. All animals were sacrificed for analysis 5 days after viral or DNA administration.Histochemical analysis and β-galactosidase assaysFor detection of β-galactosidase activity in muscles, whole tibialis anterior muscle was fixed for 5 min in 3.7% formaldehyde in PBS, cut into several pieces no more than 3 mm thick, rinsed with PBS, and stained for 12 h at 37°C with X-gal (0.1% 5-bromo-4-chloro-3-indolyl β-D-galactoside in 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2). Samples were then fixed overnight in 3.7% formaldehyde and 0.5% glutaraldehyde, embedded in glycomethacrylate, sectioned at 4-μm thickness, and counterstained with hematoxylin and eosin. For detection of β-galactosidase activity in liver, excised tissue was snap frozen in liquid nitrogen-cooled isopentane and cryosectioned at 7-μm thickness. Sections were fixed for 5 min at room temperature in 0.5% glutaraldehyde in PBS, then stained with X-gal. Protein extracts for enzymatic analysis were prepared by homogenizing flash-frozen tissue in 10 vol RIPA solution (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.1% SDS, 0.5% deoxycholate) modified by the addition of 5 mM EDTA, 1 mM Pefabloc SC, 0.5 μg/ml leupeptin, 2 μg/ml aprotinin, and 0.7 μg/ml pepstatin (38Phelps S.F. et al.Expression of full-length and truncated dystrophin minigenes in transgenic mdx mice.Hum. Mol. Genet. 1