Design of Tissue-specific Regulatory Cassettes for High-level rAAV-mediated Expression in Skeletal and Cardiac Muscle
Authors: Maja Z Salva, Charis L Himeda, Phillip WL Tai, Eiko Nishiuchi, Paul Gregorevic, James M Allen, Eric E Finn, Quynh G Nguyen, Michael J Blankinship, Leonard Meuse, Jeffrey S Chamberlain, Stephen D Hauschka
Journal: Molecular Therapy (2007)

Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and α-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is ∼400-, ∼50-, and ∼10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle. Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and α-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is ∼400-, ∼50-, and ∼10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle. IntroductionGene therapy for muscular dystrophies poses a dual challenge of efficient gene delivery to the body's largest tissue mass and selective therapeutic product expression in a physiologically diverse array of striated muscles. Advances in obtaining widespread muscle transduction in animal disease models have been achieved following systemic delivery of recombinant adeno-associated virus (rAAV) vectors.1Gregorevic P et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (539) Google Scholar, 2Mah C et al.Sustained correction of glycogen storage disease type II using adeno-associated serotype 1 vectors.Gene Ther. 2005; 12: 1405-1409Crossref PubMed Scopus (59) Google Scholar, 3Wang Z et al.Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.Nat Biotech. 2005; 23: 321-328Crossref PubMed Scopus (495) Google Scholar, 4Zhu T et al.Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer.Circulation. 2005; 112: 2650-2659Crossref PubMed Scopus (79) Google Scholar rAAV vectors are well suited for this application because serotypes 1, 6, 7, 8, and 9 efficiently transduce both skeletal and cardiac muscle, and mediate therapeutic levels of transgene expression when administered at high titers without significant pathogenic or immunogenic effects.3Wang Z et al.Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.Nat Biotech. 2005; 23: 321-328Crossref PubMed Scopus (495) Google Scholar, 4Zhu T et al.Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer.Circulation. 2005; 112: 2650-2659Crossref PubMed Scopus (79) Google Scholar, 5Kessler PD et al.Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.Proc Natl Acad Sci USA. 1996; 93: 14082-14087Crossref PubMed Scopus (533) Google Scholar, 6Xiao X Li J Samulski RJ 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, 7Herzog RW et al.Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.Nat Med. 1999; 5: 56-63Crossref PubMed Scopus (468) Google Scholar, 8Chao H Liu Y Rabinowitz J Li C Samulski RJ Walsh CE Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.Mol Ther. 2000; 2: 619-623Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 9Blankinship MJ et al.Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6.Mol Ther. 2004; 10: 671-678Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 10Sun B et al.Correction of glycogen storage disease type II by and adeno-associated virus vector containing a muscle-specific promoter.Mol Ther. 2005; 11: 889-898Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 11Inagaki K et al.Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8.Mol Ther. 2006; 14: 45-53Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 12Pacak CA et al.Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo.Circ Res. 2006; 99: E3-E9Crossref PubMed Scopus (308) Google Scholar However, widespread dissemination of systemically delivered rAAV vectors raises concerns regarding transduction of non-muscle tissues. Our group and others have shown that vectors containing muscle-specific regulatory cassettes made from enhancer and promoter portions of muscle genes, creatine kinase (CK),1Gregorevic P et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (539) Google Scholar, 4Zhu T et al.Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer.Circulation. 2005; 112: 2650-2659Crossref PubMed Scopus (79) Google Scholar, 13Hauser MA et al.Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.Mol Ther. 2000; 2: 16-25Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 14Cordier L et al.Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.Hum Gene Ther. 2001; 12: 205-215Crossref PubMed Scopus (128) Google Scholar, 15Yuasa K et al.Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscle evokes enhanced immune response against the transgene product.Gene Ther. 2002; 9: 1576-1588Crossref PubMed Scopus (111) Google Scholar, 16Jiang Z Schiedner G Gilchrist SC Kochanek S Clemens PR CTLS4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle.Gene Ther. 2004; 11: 1453-1461Crossref PubMed Scopus (22) Google Scholar, 17Yoshimura M et al.AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype.Mol Ther. 2004; 10: 821-828Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar skeletal actin,18Hagstrom JN et al.Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter.Blood. 2000; 95: 2536-2542PubMed Google Scholar myosin heavy chain (MyHC),19Skarli M Kiri A Vrbova G Lee CA Goldspink G Myosin regulatory elements as vectors for gene transfer by intramuscular injection.Gene Ther. 1998; 5: 514-520Crossref PubMed Scopus (20) Google Scholar or muscle gene control elements,20Li X Eastman EM Schwartz RJ Draghia-Akli R Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences.Nat Biotech. 1999; 17: 241-245Crossref PubMed Scopus (191) Google Scholar exhibit much lower expression in non-muscle tissues. This strategy also decreases immune responses to the transgene.10Sun B et al.Correction of glycogen storage disease type II by and adeno-associated virus vector containing a muscle-specific promoter.Mol Ther. 2005; 11: 889-898Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 13Hauser MA et al.Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.Mol Ther. 2000; 2: 16-25Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 14Cordier L et al.Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies.Hum Gene Ther. 2001; 12: 205-215Crossref PubMed Scopus (128) Google Scholar, 21Hartigan-O'Connor D Kirk CJ Crawford R Mule JJ Chamberlain JS Immune evasion by muscle-specific gene expression in dystrophic muscle.Mol Ther. 2001; 4: 525-533Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar However, in most cases such cassettes lack high-level expression in one or more striated muscle types, or their size exceeds that required for packaging within rAAV in conjunction with complementary DNAs (cDNAs) as large as the 3.7-kb microdystrophin. A possible exception is the synthetic "C5-12" muscle enhancer/promoter of about 365 bp that appears to be as active in skeletal muscle tissues and cell cultures as cytomegalovirus (CMV) cassettes.20Li X Eastman EM Schwartz RJ Draghia-Akli R Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences.Nat Biotech. 1999; 17: 241-245Crossref PubMed Scopus (191) Google Scholar, 22Liu Y-L et al.Therapeutic levels of factor IX expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector.Hum Gene Ther. 2004; 15: 783-792Crossref PubMed Scopus (45) Google Scholar, 23Gonin P Arandel L Van Wittenberghe L Marais T Perez N Danos O Femoral intra-arterial injection: a tool to deliver and assess recombinant AAV constructs in rodents whole hind limb.J Gene Med. 2005; 7: 782-791Crossref PubMed Scopus (24) Google Scholar However, the transcriptional activity of C5-12 in cardiac muscle and within different anatomical muscles and fiber types has not been studied quantitatively.4Zhu T et al.Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer.Circulation. 2005; 112: 2650-2659Crossref PubMed Scopus (79) Google ScholarPrevious work from our lab has shown that regulatory regions of the murine muscle creatine kinase (MCK) gene direct tissue-specific expression in both skeletal and cardiac muscle.24Johnson JE Wold BJ Hauschka SD 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, 25Donoviel DB Shield MA Buskin JN Haugen HS Clegg CH Hauschka SD 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, 26Shield MA Haugen HS Clegg CH Hauschka SD 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, 27Nguyen QG Buskin JN Himeda CL Shield MA Hauschka SD Differences in the function of three conserved E-boxes of the muscle creatine kinase gene in cultured myocytes and in transgenic mouse skeletal and cardiac muscle.J Biol Chem. 2003; 278: 46494-46505Crossref PubMed Scopus (20) Google Scholar, 28Nguyen QG Buskin JN Himeda CL Fabre-Suver C Hauschka SD Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types.Transgenic Res. 2003; 12: 337-349Crossref PubMed Scopus (18) Google Scholar Based on many transgenic mouse and cell culture studies, most transcription from the endogenous MCK gene is mediated by a 206-bp enhancer, located ∼1.2 kb 5′ of the transcription start site, and a 358-bp proximal promoter (Figure 1a).26Shield MA Haugen HS Clegg CH Hauschka SD 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, 29Amacher SL Buskin JN Hauschka SD 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 However, whereas endogenous MCK transcripts are only ∼3-fold lower in cardiac than in skeletal muscle,24Johnson JE Wold BJ Hauschka SD 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 MCK transgenes containing only the enhancer and promoter (CK1 cassette; see Figure 1a) typically exhibit only ∼1% as much activity in the heart as in skeletal muscle.26Shield MA Haugen HS Clegg CH Hauschka SD 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, 28Nguyen QG Buskin JN Himeda CL Fabre-Suver C Hauschka SD Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types.Transgenic Res. 2003; 12: 337-349Crossref PubMed Scopus (18) Google Scholar In earlier studies, modifying the enhancer portion of the CK1 cassette at two sites (Figure 1c, CK6) yielded high expression in muscle cultures and potentially therapeutic levels of microdystrophin expression in the skeletal muscles of mdx mice, but very poor expression in cardiac muscle.1Gregorevic P et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (539) Google Scholar, 13Hauser MA et al.Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.Mol Ther. 2000; 2: 16-25Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 30Scott JM et al.Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin.Neuromuscul Disord. 2002; 12 (Suppl): S23-S29Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar As death in Duchenne muscular dystrophy patients is often due to heart failure, it is important to achieve robust transgene expression in cardiac muscle. To this end, we have designed new cassettes that drive high-level tissue-specific transgene expression in both cardiac and skeletal muscle, and which meet the packaging size constraint (maximum size 800 bp) for accommodating the microdystrophin cDNA in rAAV vectors.ResultsCassette designThe CK6 cassette (Figure 1c) was useful in early rAAV-microdystrophin studies owing to its small size (610 bp) and robust performance in skeletal muscle,30Scott JM et al.Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin.Neuromuscul Disord. 2002; 12 (Suppl): S23-S29Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar but its activity in cardiac muscle was low. To rectify CK6's low activity in cardiac muscle, the native mouse CK1 cassette was used as the starting point for new modifications (Figure 1a). The CK7 cassette incorporates three successive modifications, each of which increased activity (data not shown). The first change is a 63-bp deletion between the enhancer right E-box and MEF2 sites which eliminates a stretch of poorly conserved sequence (Figure 1a), and that brings the right E-box and MEF2 sites into close proximity, thus facilitating positive interactions between myogenic regulatory factors (MRFs) transcription factors bound to the E-box and MEF2 transcription factors bound to the MEF2 site.31Black BL Molkentin JD Olson EN Multiple roles for the MyoD basic region in transmission of transcriptional activation signals and interaction with MEF2.Mol Cell Biol. 1998; 18: 69-77Crossref PubMed Scopus (126) Google Scholar The second modification changes the sequence overlapping the transcription start site to as consensus initiator sequence (Inr) that synergizes with the TATA box by increasing the binding affinity of initiation factors IID and IIA.32Emami KH Jain A Smale ST Mechanism of synergy between TATA and initiator: synergistic binding of TFIID following a putative TFIIA-induced isomerization.Genes Dev. 1997; 11: 3007-3019Crossref PubMed Scopus (80) Google Scholar The third modification was inclusion of a highly conserved 50 bp from the MCK exon-1 5′-UTR. In addition to increasing cassette activity by ∼2-fold in MM14 skeletal myocytes, this modification also led to an ∼5-fold increase in activity in neonatal rat cardiomyocytes.As some vertebrate genes contain multiple enhancers,33Wade DP Puckley LH Knight BL Acquati F Mihalich A Taramelli R Characterization of multiple enhancer regions upstream of the apolipoprotein(a) gene.J Biol Chem. 1997; 272: 30387-30399Crossref PubMed Scopus (54) Google Scholar it was possible that addition of a strong cardiac muscle gene enhancer to the CK7 cassette would augment transcription in heart muscle without perturbing the cassette's activity in skeletal muscle. As the 188-bp mouse α-myosin heavy chain complex (α-MHC) enhancer (Figure 1b) is the shortest enhancer known to confer high-level cardiac muscle-specific activity in the context of a heterologous promoter,34Molkentin JD Jobe SM Markham BE α-Myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter.J Mol Cell Cardiol. 1996; 28: 1211-1225Abstract Full Text PDF PubMed Scopus (54) Google Scholar it was ligated to the CK7 cassette in positive and negative orientations to create the 770-bp MHCK7 cassettes. To ascertain how the α-MHC enhancer functions with native MCK control elements, the α-MHC enhancer was also ligated to the CK1 cassette in either orientation to create the MHCK1 cassettes. All regulatory cassettes were cloned upstream of the human placental alkaline phosphatase (AP) reporter gene and into constructs containing AAV2 inverted terminal repeats for packaging into rAAV vectors (Figure 1d).Cassette evaluation in vitroCassettes were first evaluated following transient transfection of MM14 skeletal muscle cells (Figure 2a). As expected, the CK6 cassette had ∼2-fold higher relative activity than the "native" CK1 cassette. Importantly, the modifications incorporated into the CK7 cassette resulted in a threefold increase in activity relative to the CK1 cassette, and addition of the α-MHC enhancer to the CK7 cassette resulted in activity levels ∼7- to 10-fold higher than the CK1 cassette and comparable to those of the CMV and Rous sarcoma virus (RSV) viral promoters (Figure 2a).Figure 2Cassette activity in vitro. (a) Proliferating MM14 mouse muscle cells were co-transfected with a test plasmid expressing AP under control of the indicated cassette and a reference plasmid expressing β-galactosidase under control of the −1256/+7 region of the MCK promoter, and switched to differentiation media 4 h later. (b) HEK 293 cells, a human fibroblast cell line, (c) L cells, a mouse fibroblast cell line, and (d) JAWSII mouse immature dendritic cells were transfected with the same test plasmids as MM14 cells and a reference plasmid expressing β-galactosidase under control of the CMV promoter. Cell lysates were harvested 48 h (MM14, HEK 293, and L cells) or 24 h (JAWSII cells) after transfection and AP and β-galactosidase activity were measured by luminometric assays. Activity is shown as a percentage of CMV activity. Error bars represent SDs, n=6 (MM14 myocytes) or 3 (non-muscle cells).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cassette activity was also evaluated in a human fibroblast embryonic kidney cell line (HEK 293 cells), a mouse fibroblast cell line (L cells), and a mouse immature dendritic cell line (JAWSII) to assess the extent of muscle-specific expression (Figure 2b–d). The activity of CK1-based cassettes in both fibroblast cell lines was very low, about 1% of CMV activity. CK7-based cassettes were slightly more active, but these cassettes were very weak compared with CMV, thus confirming retention of their muscle specificity. In contrast, the activity of the RSV promoter was ∼50% of CMV in 293 cells and ∼15% in L cells. Importantly, none of the MCK-based cassettes displayed activity levels above background in JAWSII dendritic cells, whereas the CMV promoter was very active in these cells.Systemic delivery of rAAV6 vectorsTo test cassette activity in vivo, the constructs were packaged into pseudotype rAAV6 vectors (AAV2 genomes packaged in AAV6 capsids) and 6 × 1011 vector genomes were delivered systemically by tail vein injection in mice. This dose was previously shown by us and others1Gregorevic P et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (539) Google Scholar to achieve sufficiently high transduction levels to detect cassette activity in all muscles while not saturating the AP levels detected in tissue sections.Analysis of vector genome distribution showed that all tissues contained viral genomes at the 4-week time of harvest (Figure 4a). As previously seen, the liver had the highest concentration of viral genomes; this is due to the route of administration.1Gregorevic P et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (539) Google Scholar, 35Grimm D et al.Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy.Blood. 2003; 102: 2412-2419Crossref PubMed Scopus (185) Google Scholar The soleus and heart had two- to fivefold lower vector genome levels, other muscles and organs had 10- to 100-fold lower levels than liver, and muscles such as the quadriceps and diaphragm had nearly 10-fold lower vector numbers than the soleus. Most organs had similar concentrations of viral genomes as the quadriceps muscle. Whereas vector numbers in Figure 4a are based on data for eight different cassettes in four mice each, we detected considerable mouse-to-mouse variability (see error bars), as well as between the averages for each cassette (e.g., note that the −MHCK7 cassette has the lowest viral genome number in almost every muscle and tissue). With these variations in mind, normalizing regulatory cassette activity to viral genome numbers should compensate for at least some vector number differences. This analysis does not, however, disclose what cell types within each tissue contain the genomes. Thus, when the vector genome quantities are used to normalize regulatory cassette expression from one muscle or tissue to another, the implicit, but unproven, assumption is that the vector genomes are distributed in equivalent proportions and with equivalent potential functionality within each cell type.Figure 4Quantitative analysis of AP expression following systemic delivery of rAAV6 vectors. A total of 6 × 1011 vector genomes of rAAV6 vectors encoding AP driven by the indicated cassettes (key shown in (a) is used throughout the entire figure) were administered by tail vein injection and tissues were harvested 4 weeks later. (a) Viral genome distribution in muscles and non-muscle tissues. Total DNA was extracted from each tissue and the relative number of vector genomes was determined by quantitative PCR using plasmid DNA as a standard curve. Data are expressed as vector genomes per nanogram of total DNA. Error bars represent SDs, n=4. (b) Cassette activity in skeletal muscles. Muscles from one side were used for tissue lysate preparation and genome extraction, whereas muscles from the contralateral side were used for histology. AP activities of tissue lysates of soleus muscle, diaphragm, quadriceps muscle, and gastrocnemius muscle are shown after subtracting the activity from uninjected tissue lysates, normalized to total protein and number of vector genome copies. Error bars represent SDs, n=4. (c) Cassette activity in non-muscle tissues. AP activities in tissue lysates of liver, spleen, lung, intestine, and testes are shown after subtracting the activities of tissue lysates from uninjected mice. All values are normalized to total protein and number of vector genome copies. Error bars represent SDs, n=4. Activities in kidney, aorta, and brain lysates were at background level for all constructs (data not shown). Background level activity in (b and c) is indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cassette activity in cardiac muscleCassettes were first evaluated in cardiac muscle by measuring AP activity in tissue lysates (Figure 3a). As anticipated, the CK6 cassette was weakest in cardiac muscle, with ∼6-fold lower activity than the native CK1 cassette. Addition of the α-MHC enhancer to CK1 increased the activity by about sixfold. The CK7 cassette performed even better, with ∼16-fold increase over CK1, and the MHCK7 cassettes exhibited the highest overall activities, although not significantly different from those of CK7, CMV, and RSV. To assess cassette activity in single cells, frozen sections were stained for AP activity (Figure 3b). Only ∼2% of the cells were AP-positive in sections from mice injected with the CK6 vectors, and this correlates well with low AP activity in heart lysates from the same mice. Sections from CK1-injected mice had more positive cells, but expression was mosaic with many cells showing little or no activity. Sections from MHCK1-injected mice also exhibited mosaic expression, but more cells were AP-positive, again correlating well with luminometry data. Finally, CK7 and MHCK7 as well as the viral cassettes all drive high-level expression in cardiac muscle, with nearly 100% of the cells appearing AP-positive.Figure 3Cassette activity in cardiac muscle. A total of 6 × 1011 vector genomes of rAAV6 vectors were administered by tail vein injection and hearts were harvested 4 weeks later for assaying. Each heart was subdivided into three portions, one for tissue lysate preparation, one for DNA extraction, and one for histology. (a) AP activity of cardiac tissue lysates is shown after subtracting activity of uninjected tissue lysates, normalized to total protein and number of vector genome copies. Error bars represent SDs, n=4. (b) AP activity in sections of cardiac muscle stained with BCIP/NBT substrate for 1 h. Representative images obtained using a 10 × objective are shown. Bar=200 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cassette activity in skeletal musclesCassette activity was assessed in skeletal muscles with different functions and fiber type compositions. Data for diaphragm (an essential respiratory muscle containing fast glycolytic type IId/x and slow type I fibers), soleus (a leg muscle containing ∼45% slow type I and fast oxidative type IIa fibers), quadriceps, and gastrocnemius (leg muscles containing ⩾80% fast glycolytic type IIb fibers) are presented in Figures 4b and 5.36Allen DL Harrison BC Sartorius C Byrnes WC Leinwand LA Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy.Am J Physiol Cell Physiol. 2001; 280: C637-C645PubMed Google Scholar In the diaphragm, CK1 cassette activity was very low and CK6 activity was undetectable (Figure 4b). Addition of the α-MHC enhancer increased the activity ∼4-fold, whereas the CK7 modifications provided a 13-fold increase, relative to CK1. MHCK7 performed best in the diaphragm, with the + and − orientations of the α-MHC enhancer providing 18- and 36-fold greater activity than CK1, although the viral promoters were slightly more active. AP staining in the diaphragm (Figure 5a) confirmed that CK1, CK6, and MHCK1 express poorly, whereas CK7 and MHCK7 cassettes were express