Enhancer Drives Expression of Mini-dystrophin in Transgenic mdx Mice and Rescues the Dystrophy in These Mice

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doi:10.1016/j.ymthe.2006.04.013

The Mouse Dystrophin Muscle Promoter/Enhancer Drives Expression of Mini-dystrophin in Transgenic mdx Mice and Rescues the Dystrophy in These Mice Carrie L. Anderson,1,2 Yves De Repentigny,1,2 Carlo Cifelli,3 Philip Marshall,1,2 Jean-Marc Renaud,3 Ronald G. Worton,1,2,4,5 and Rashmi Kothary1,2,3,5,* 1 Ottawa Health Research Institute, Ottawa, ON, Canada K1H 8L6 Center for Neuromuscular Disease, 3Department of Cellular and Molecular Medicine, 4Department of Biochemistry, Microbiology, and Immunology, and 5Department of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 2

*To whom correspondence and reprint requests should be addressed at the Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON, Canada K1H 8L6. Fax: +1 613 737 8803. E-mail: [email protected].

Available online 27 June 2006

Successful gene therapy for Duchenne muscular dystrophy (DMD) requires the restoration of dystrophin protein in skeletal muscles. To achieve this goal, appropriate regulatory elements that impart tissue-specific transgene expression need to be identified. Currently, most muscle-directed gene therapy studies utilize the muscle creatine kinase promoter. We have previously described a muscle enhancer element (mDME-1) derived from the mouse dystrophin gene that increases transcription from the mouse dystrophin muscle promoter. Here, we explore the use of this native mouse dystrophin muscle promoter/enhancer to drive expression of a human dystrophin minigene in transgenic mice. We show that the dystrophin promoter can provide tissue-specific transgene expression and that the mini-dystrophin protein is expressed at the sarcolemma of skeletal muscles from mdx mice, where it restores the dystrophin-associated glycoprotein complex. The level of transgene expression obtained is sufficient to protect mdx muscles from the morphological and physiological symptoms of muscular dystrophy, as well as from exercise-induced damage. Therefore, the dystrophin muscle promoter/enhancer sequence represents an alternative for use in gene therapy vectors for the treatment of DMD. Key Words: gene therapy, DMD, muscular dystrophy, transgenic rescue, dystrophin muscle promoter, MCK promoter

INTRODUCTION Duchenne and Becker muscular dystrophies (DMD/BMD) are X-chromosome-linked recessive muscle wasting diseases that are caused by defective expression of dystrophin [1,2]. The dystrophin gene spans 2.9 Mb. It consists of 79 exons and is expressed as a 14-kb transcript in muscle cells [3,4]. A mouse model for DMD is the mdx mouse [5], which features a point mutation in exon 23 of the dystrophin gene [6]. Although the mdx mouse does not display any overt signs of muscle weakness or movement difficulty, its phenotype is characterized by cycles of muscle degeneration/regeneration. Replacement of dystrophin by gene therapy is one strategy to slow the progression of DMD but the large size of the gene and corresponding mRNA makes development of such strategies a daunting challenge (reviewed in [7]). However, work by Chamberlain and others has

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shown that expression of dystrophin mini- and microgenes may be a viable option for the treatment of DMD [8–17]. Identification of promoters that ensure appropriate tissue-specific expression of the therapeutic gene is thus important for achieving gene therapy for DMD. Previous efforts have utilized dystrophin mini- and microgenes under the control of the muscle creatine kinase (MCK) gene promoter/enhancer to achieve muscle-specific expression [8,10,15,18]. The MCK regulatory elements have been well characterized, both in cell culture and in transgenic mice [8,19–21]. Initially characterized as a 6.5-kb promoter/enhancer segment, smaller versions also function as muscle-specific regulatory elements, including for example a 1.35-kb MCK promoter/enhancer element driving the expression of a mini-dystrophin gene in transgenic mice [8]. With this promoter, the muscular dystrophy phenotype was atte-

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nuated in fast, but not slow, muscles of transgenic mdx mice. However, this latter study was with a single line of transgenic mice and therefore the fiber-type specificity may have been due to the site of transgene integration. Overall, although the MCK promoter has shown great promise in its use as a muscle-specific regulatory element, the dystrophin gene promoter also deserves consideration. To date there have been eight promoters identified at the DMD locus producing eight different tissue-specific isoforms (reviewed in [22]). Of these, only the muscle, brain, and cerebellar Purkinje promoters produce transcripts encoding a full-length 427-kDa dystrophin protein [23–27]. The first 150 nucleotides of the muscle promoter are required for muscle-specific expression in cultured cells [28]. A transcriptional enhancer (dystrophin muscle enhancer-1) has been identified 6.5 kb downstream of the muscle promoter within muscle intron 1 of the human dystrophin gene [29]. This enhancer increases transcription from the dystrophin muscle promoter in both myoblasts and myotubes [30]. Likewise, we characterized an intron-1 enhancer element (mouse dystrophin muscle enhancer-1; mDME-1) 8.5 kb downstream of the mouse dystrophin muscle promoter [31]. A 3-kb fragment harboring mDME-1 increased transcription from the mouse dystrophin muscle promoter in cultured myotubes. In transgenic mice, a mouse dystrophin muscle promoter/enhancer–lacZ transgene was expressed in both skeletal muscle and compartments of the heart [32]. Thus, the dystrophin muscle promoter is dependent on the enhancer sequence to target both skeletal and heart muscle. To determine whether the mouse dystrophin muscle promoter/enhancer has any therapeutic potential, we have generated transgenic mice expressing the human dystrophin minigene. We demonstrate that this promoter/enhancer combination is capable of imparting transgene expression to skeletal muscle and that this is sufficient to restore the wild-type phenotype in mdx mice. Muscles from the transgenic mdx mice are protected against exercise-induced damage and are not morphologically or physiologically different from their wild-type littermates. This work highlights the potential of using dystrophinTs own regulatory elements in gene therapy for DMD.

RESULTS Generation of Dystrophin Muscle Promoter/Enhancer Dystrophin Minigene Transgenic Mice We have used our previously characterized mouse dystrophin muscle promoter/enhancer cassette [31,32] to drive muscle-specific expression of the human dystrophin minigene (Fig. 1A) in transgenic mice. We obtained four founder mice and our initial analysis was directed at examining the pattern of expression of the dystrophin

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minigene transcript in two separate transgenic lines (Tg1888 and Tg1867). We demonstrate by reverse-transcriptase polymerase chain reaction (RT-PCR) that the transgene is expressed in several different muscle types in line Tg1888 (Fig. 1B, top). Interestingly, the transgene is also expressed in brain and at a reduced level in the heart of these mice. By contrast, it is not expressed in the liver and kidney (Fig. 1B). The expression of the transgene in the second line, Tg1867, was considerably restricted and at a reduced level (Fig. 1B, bottom). This variability in transgene expression likely reflects the influence of site of integration. Tissue-Specific Expression of the Dystrophin Minigene Previous work has shown that the expression of the fulllength 427-kDa dystrophin protein in normal wild-type mice is restricted to skeletal muscle, heart, and brain. We performed an immunoblot analysis of protein extracts from various tissues of wild-type or Tg1888 mice and have confirmed these observations (Fig. 1C). The fulllength dystrophin protein is detected in several muscle groups and in heart. It is also present in brain at reduced levels. As expected, the endogenous dystrophin protein is not detected in liver and kidney (Fig. 1C). We performed further immunoblot assays and, consistent with the RT-PCR analysis, the transgene product is present in several muscle groups of line Tg1888 (Fig. 1C). The mini-dystrophin protein is also detected in brain, but not in liver and kidney. We were unable to detect transgene-derived mini-dystrophin in tissues from the second line, Tg1867 (data not shown). We used strain Tg1888 for all subsequent experiments presented in this paper. The transgene was bred onto the mdx background by crossing Tg1888 male mice with mdx females. Male offspring were screened for the presence of the transgene to distinguish between mdx and mdx/tg mice. Analysis of protein extracts from tibialis anterior (TA) muscle reveals the presence of the full-length dystrophin protein in the wild-type mice and in the Tg1888 mice, but not in the mdx and mdx/tg mice (Fig. 1D). As expected, the mini-dystrophin protein is expressed only in the Tg1888 and mdx/tg mice (Fig. 1D), and the level of expression of mini-dystrophin is substantially greater in the mdx background, a consistent observation in this study (e.g., see Fig. 2H). Immunofluorescence analysis of the TA muscles from wild-type, mdx, and mdx/tg mice revealed the expected sarcolemmal distribution of full-length dystrophin in the wild-type muscle (Fig. 2A) but its absence in the mdx muscle (Fig. 2B). Parallel analysis of TA muscle sections from mdx/tg mice demonstrates sarcolemmal localization of the mini-dystrophin protein (Fig. 2C). The level of sarcolemmal mini-dystrophin in mdx/tg muscle was lower compared with sarcolemmal dystrophin in wildtype muscle. However, from our examination of several

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FIG. 1. Derivation of transgenic mice and expression analysis. (A) Schematic representations of dystrophin and related constructs. Structural domains of fulllength dystrophin, mini-dystrophin, and the mouse dystrophin muscle promoter/muscle enhancer mini-dystrophin transgene construct are shown. The fulllength dystrophin consists of an N-terminal actin-binding domain (ABD), four hinge regions (H1–H4), 24 spectrin-like repeats (R1–R24), a cysteine-rich domain that binds to h-dystroglycan, and a C-terminal domain that binds to syntrophins and dystrobrevins. In our minigene, the rod domain is disrupted by deletion of hinge region 2 and repeats 4–19 (DH2-R19), corresponding to deletion of exons 17–48. The transgene construct has a mouse dystrophin muscle enhancer (mDME-1), muscle-specific dystrophin promoter (MP), and SV40 polyadenylation signal controlling the expression of the mini-dystrophin cDNA. (B) RT-PCR analysis of transgene expression in lines Tg1888 (top) and Tg1867 (bottom). The tissues examined are heart, tibialis anterior (TA) muscle, diaphragm (DIAPH), kidney, liver, gastrocnemius (GAST) muscle, brain, biceps femoral (BF) muscle, soleus (SOL) muscle, and extensor digitorum longus (EDL) muscle. The negative control (neg CTL) consisted of no RNA in the RT reaction. All tissues shown are transgenic except for TA wt. (C) Immunoblot analysis of protein extracts (50 Ag) from various tissues of a 4-week-old B6C3F1/tg mouse and 9-week-old B6C3F1 wild-type (wt) mouse. All tissues shown are transgenic except for TA wt. Fulllength dystrophin (dys) is readily detected in heart, brain, tibialis anterior (TA) muscle, biceps femoral (BF) muscle, lateral gastrocnemius (LG) muscle, and medial gastrocnemius (MG) muscle. In contrast, the endogenous protein is not detectable in liver and kidney. By comparison, the minigene product is detected in brain, TA, LG, and MG muscles, but not in heart, liver, kidney, or BF muscle. (D) Immunoblot analysis of protein extracts (50 Ag) from the TA muscle of 6-week-old male B6C3F1 (wild type), B6C3F1/tg (transgenic), mdx, and mdx/tg mice. Endogenous full-length dystrophin is detected in B6C3F1 and B6C3F1/tg muscles only. Correspondingly, the mini-dystrophin product is detected in the skeletal muscle of B6C3F1/tg mice and at elevated levels in mdx/tg mice.

samples, virtually every muscle fiber of the mdx/tg background was dystrophin positive. We counted the number of total strongly dystrophin positive muscle

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fibers in four different mdx/tg mice. We made the assumption that btotal strongly positiveQ fibers should require that at least 95% of the perimeter of the fiber be

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FIG. 2. Expression analysis of dystrophin and mini-dystrophin in control and transgenic mdx muscles. Tibialis anterior muscle tissue from 2.5-month-old (A) B6C3F1 wild-type, (B) mdx, and (C) mdx/tg mice was sectioned transversally and immunostained with dystrophin antisera. Staining of mdx TA muscle reveals the absence of dystrophin (B), whereas control TA muscle sections demonstrate normal sarcolemmal localization of dystrophin (A). Parallel analysis of TA muscle sections from mdx/tg mice demonstrates sarcolemmal localization of the mini-dystrophin protein (C). Note the regularity of dystrophin expression in both the wild-type and the mdx/tg muscles. (D) The secondary antibody control. Scale bar, 50 Am. Diaphragms from 10-week-old (E) B6C3F1 wild-type, (F) mdx, and (G) mdx/tg mice were cross-sectioned and immunostained with dystrophin antisera. Control diaphragm muscle sections demonstrate normal sarcolemmal localization of dystrophin (E), whereas staining of mdx diaphragm muscle reveals the absence of dystrophin (F). (G) Diaphragm muscle sections from mdx/tg mice demonstrate sarcolemmal localization of the mini-dystrophin protein in many but not all fibers. Scale bar, 50 Am. (H) Immunoblot analysis of proteins (50 Ag) extracted from the TA and diaphragm muscle of 9-week-old male B6C3F1 (wild type), B6C3F1/tg (transgenic), mdx, and mdx/tg mice. Endogenous fulllength dystrophin is detected in both TA muscle and diaphragm of B6C3F1 and B6C3F1/tg mice. Mini-dystrophin is detected in both TA muscle and diaphragm of B6C3F1/tg and mdx/tg mice. Note that expression of mini-dystrophin in mdx/tg diaphragm is lower than that in mdx/tg TA muscle.

very bright. We measured the percentage of total strongly positive fibers and the numbers were consistent in all four mice (27, 26.6, 27.6, and 27.5%). We next examined the diaphragm of the mdx/tg mice for expression of the mini-dystrophin protein. Staining of control diaphragm muscle sections demonstrates normal

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sarcolemmal localization of dystrophin, whereas staining of the mdx diaphragm muscle reveals the absence of dystrophin (Figs. 2E and 2F). Parallel analysis of diaphragm muscle sections from mdx/tg mice demonstrates that the minigene is expressed and that the minidystrophin protein is localized at the sarcolemma (Fig.

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2G). Curiously, not all fibers within the diaphragm express mini-dystrophin at the sarcolemma. Furthermore, immunoblot analysis of proteins extracted from the diaphragm muscle reveals that although the minidystrophin protein is detectable in mdx/tg mice, it is at a level much lower than that in the TA muscle of the same mouse (Fig. 2H). Restoration of the DAG Complex in the mdx/tg Mice Ablation of dystrophin or members of the dystrophinassociated glycoprotein (DAG) complex generally results in a down-regulation of other members of this complex. We performed a study on how the distribution of the DAG complex is affected in mdx and mdx/tg skeletal muscle. We sectioned TA muscles from 3-week-old mice and immunostained them with antisera against dystrophin, hdystroglycan, and g-sarcoglycan (Fig. 3). As expected, the DAG complex protein distribution is normal in wild-type muscle and localization to the sarcolemma is observed (Figs. 3A, 3D, and 3G). In contrast, dystrophin, as well as h-

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dystroglycan and g-sarcoglycan, is either absent or severely diminished in the sarcolemma of mdx muscle (Figs. 3B, 3E, and 3H). The loss of the DAG complex from the sarcolemma of mdx muscle is corrected by the expression of the mini-dystrophin protein in the mdx/tg muscle (Figs. 3C, 3F, and 3I). Thus, the level of expression of the minigene from the dystrophin muscle promoter/ enhancer, although low, is sufficient to ensure restoration of the DAG complex. Histopathological Analysis of Skeletal Muscle We performed histopathology of hind-limb skeletal muscle from 4-week-old mice to determine whether expression of mini-dystrophin protein from the dystrophin muscle promoter/enhancer was sufficient to prevent pathology. Hematoxylin–eosin stained cross sections of wild-type and transgenic TA muscle have normal morphology, with consistency in fiber caliber and minimal evidence of central nuclei (Figs. 4A and 4B). In contrast, staining of mdx TA muscle sections demonstrates mor-

FIG. 3. Restoration of the dystrophin-associated glycoprotein (DAG) complex in mdx/tg mice. Tibialis anterior muscles from 3-week-old (A, D, and G) B6C3F1 wild-type, (B, E, and H) mdx, and (C, F, and I) mdx/tg mice were collected and sections stained with antisera against the following members of the DAG complex: dystrophin (DYS), h-dystroglycan (h-DG), and g-sarcoglycan (GSG). Staining of mdx TA muscle reveals the absence of DAG complex proteins from the sarcolemma (B, E, and H), whereas control TA muscle sections demonstrate normal sarcolemmal localization of these proteins (A, D, and G). Parallel analysis of TA muscle sections from mdx/tg mice demonstrates restoration of the DAG complex proteins to the sarcolemma (C, F, and I). Scale bar, 50 Am.

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FIG. 4. H&E staining of control and transgenic mdx muscles. Sections of tibialis anterior muscles from 4-week-old (A) B6C3F1 wild-type, (B) B6C3F1/tg, (C) mdx, and (D) mdx/tg mice are shown. (A and B) Control and transgenic TA muscle sections demonstrate normal morphology with consistency in fiber caliber and minimal evidence of central nuclei. In (E) cross-sectional area of individual fibers was determined and results are presented as a bar graph; 300 fibers each of B6C3F1 and B6C3F1/tg sections were measured. (C) H&E staining of mdx TA muscle sections demonstrates morphological characteristics of dystrophy, including variation in fiber size, mononuclear cell infiltrates, fibrosis, and abundant centrally located myonuclei. (D) Parallel analysis of TA muscle sections from mdx/tg mice demonstrates a broader distribution of fiber size with fewer central nuclei and healthier looking fibers. (F) Once again, cross-sectional area of individual fibers was determined (300 mdx fibers and 268 mdx/tg fibers), and the results are presented as a bar graph. There are greater numbers of small-caliber fibers in the mdx muscle, whereas the mdx/tg muscles have more large fibers. Scale bars in A, B, C, and D, 50 Am.

phological characteristics of dystrophy, including variation in fiber size, mononuclear cell infiltrates, fibrosis, and abundant centrally located myonuclei (Fig. 4C). Conversely, parallel analysis of TA muscle sections from mdx/tg mice demonstrates a broader range of fiber size with fewer central nuclei and healthier looking fibers (Fig. 4D). Quantification of fiber cross-sectional areas revealed that the fibers (n = 300 fibers) in sections of muscle from wild-type or transgenic mice have a normal distribution

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pattern (Fig. 4E). Similarly, we determined the crosssectional area of individual fibers for mdx (n = 300 fibers) and mdx/tg (n = 268 fibers) muscle, and the results indicated that there are greater numbers of small-caliber fibers in the mdx muscle, whereas the mdx/tg muscles have more large fibers (Fig. 4F). We also quantified the occurrence of centrally nucleated fibers in the same TA muscle sections used for the histopathology. The muscles from wild-type and

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TABLE 1: Quantification of centrally located nuclei in TA muscle fibers of control, mdx, and mdx/tg mice Genotype

Fibers with centrally located nucleus

% Central nuclei

3 of 300 2 of 300 92 of 300 23 of 268

1 0.7 30.7 8.6

wt tg mdx mdx/tg

B6C3F1/tg mice have less than 1% centrally nucleated myofibers, whereas those from mdx mice have 30.7% centrally nucleated fibers (Table 1). Expression of the transgene in the mdx background decreased the incidence of centrally nucleated fibers to 8.6% (Table 1). Thus, the expression of the transgene from the dystrophin muscle promoter/enhancer is capable of at least partially rescuing the dystrophic phenotype in mdx muscles. Protection From Exercise-Induced Muscle Damage We next determined whether dystrophin muscle promoter/enhancer-driven mini-dystrophin expression is capable of providing protection to exercise-induced muscle damage. We put the mice either in a bnonexercisedQ sedentary group or in an exercised group, which was subjected to a regimen of running on a treadmill for 10 min/day for a period of 3 days [33]. We then visualized degenerating muscle fibers by in vivo staining with Evans blue dye. We analyzed several muscle types, and to quantify the observations better, we measured the area of muscle cross section labeled with the Evans blue dye and expressed it as a percentage of the total area. We did this for several muscles, including the TA, extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (G), which together represent the hindlimb muscles. As well, we performed analysis of the biceps brachii (BB) as representatives of the forelimb muscles. Finally, we included the diaphragm (DIA) as a respiratory muscle. The results obtained from two mice of each group are summarized in Table 2. The only damaged fibers observed in the sedentary or exercised wild-type mice were a small cluster in the soleus of one exercised wild-type mouse. In contrast, several muscles from the sedentary mdx mice displayed significant muscle damage.

After 3 days of exercise, there was a general accentuation of muscle fiber damage in some of the muscles in the mdx mice, with the most notable effect being in the TA, the BB, and the diaphragm muscles. The expression of minidystrophin reduced the occurrence of damage in the TA, EDL, SOL, G, and BB muscles of mdx/tg mice. Indeed, the amount of muscle damage in the hind-limb and forelimb muscles of sedentary mdx/tg mice approached that observed for the wild-type mice. Furthermore, the transgene appeared to provide sufficient protection from exercise-induced muscle damage for mdx/tg mice. The one exception to this was in the diaphragm, where the damage already present in sedentary mdx/tg mice was worsened after exercise, as it was in the diaphragm from mdx mice. This is consistent with our observation of mosaic expression of the transgene in the diaphragm (Fig. 2G). Taken together, our results suggest that the dystrophin muscle promoter/enhancer can drive sufficient expression of the mini-dystrophin gene to most skeletal muscle types to impart protection from exercise-induced damage. Mini-dystrophin Expression Restores Muscle Contractility in Soleus Muscle of mdx Mice To determine whether the mini-dystrophin expression in our mdx/tg mice could improve muscle contractility, we chose to examine the soleus muscle of 8-week-old mice. We obtained samples from five wild-type, mdx, and mdx/ tg mice and processed them for the force measurements as described under Materials and Methods. We first examined the contractile properties during twitch and tetanic contraction at the physiological temperature of 378C. Most of the twitch parameters were not significantly different between wild type, mdx, and mdx/tg mice (Table 3). The mean peak tetanic force was significantly less in mdx soleus compared to wild-type soleus, while the maximum rates of force development and relaxation were lower but not significantly different. All three parameters were significantly greater in mdx/tg soleus than in mdx soleus. From the force–frequency relationship between the various samples, we also observed that the peak forces of mdx soleus were significantly less than those from wild-type soleus when the stimulation frequencies were 120 Hz or greater (Fig.

TABLE 2: Distribution of damaged areas (in %) in different muscles of control, mdx, and mdx/tg mice and the effects of exercise Muscle TA EDL SOL G BB DIA

Sedentary wt 0 0 0 0 0 0

0 0 0 0 0 0

Exercised wt 0 0 0 0 0 0

0 0 0.18 0 0 0

Sedentary mdx

Exercised mdx

0 4.60 0 3.24 0.78 3.43

6.48 0 3.90 0.92 49.81 14.83

0.30 0 5.46 9.21 1.25 6.00

4.14 5.97 0 2.38 6.49 5.89

Sedentary mdx/tg 0 0 0 0 0.54 2.08

0 0 n.d. 1.49 0 4.18

Exercised mdx/tg 0 0 0 0 0.18 14.18

0 0 0 1.14 1.84 7.04

Values from two mice per category are shown in each column. n.d., not determined.

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TABLE 3: Contractile properties of control, mdx, and mdx/tg soleus muscle at 378C Parameter Twitch Peak force (N/cm2) Half-rise time (ms) Time to peak (ms) Half-relaxation time (ms) Width (ms) Maximum rate of force development (N/cm2s) Maximum rate of relaxation (N/cm2s) Tetanus (200 Hz) Peak force (N/cm2) Half-rise time (ms) Half-relaxation time (ms) Maximum rate of force development (N/cm2s) Maximum rate of relaxation (N/cm2s)

wt 2.3 2.7 9.4 10.4 17.2 378 155

F F F F F F F

mdx 0.5 0.1 0.5 0.4 0.7 92 33

32.3 F 4.9 20.0 F 1.1 45.9 F 0.7 954 F 202 1203 F 198

mdx/tg

2.8 F 0.4 2.9 F 0.2 11.4 F 0.2* 11.8 F 1.4 20.3 F 1.6 419 F 50 164 F 27

2.3 F 0.7 3.0 F 0.1 10.6 F 0.5 9.6 F 0.9 17.2 F 1.1 364 F 98 158 F 30

23.9 F 2.3* 16.3 F 0.5* 45.7 F 2.9 810 F 65 860 F 108

32.4 F 2.5** 16.0 F 0.5* 40.4 F 1.6* 1132 F 87** 1216 F 79**

Values are means F SE from five mice. 4 Significantly different from wild-type mice, t test P b 0.05. 44 Significantly different from mdx mice, t test P b 0.05.

5). However, the peak forces of mdx/tg soleus were not only significantly greater than those of mdx soleus, they were also similar to the peak forces of wild-type soleus (Fig. 5).

DISCUSSION In the present study, we have used the native dystrophin muscle promoter/enhancer to drive expression of a

FIG. 5. Functional recovery in soleus muscle of mdx/tg mice when tested in vitro. Force–frequency curves were measured by increasing the stimulation frequency from 10 to 200 Hz in 10-Hz increments (for clarity not all data are shown). *Mean peak force with significant difference from the mean peak force of wild-type muscles (ANOVA and LSD P b 0.05). §Mean peak force of mdx/tg muscle with significant difference from the mean peak force of mdx muscle (ANOVA and LSD P b 0.05).

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dystrophin minigene in transgenic mice. Analysis of two different transgenic lines revealed that this promoter/enhancer sequence has the capability to drive transgene expression to muscle tissues. However, there was variability in expression, and line Tg1888 displayed a broader range of tissue expression than did line Tg1867. This difference is likely due to an influence of the site of transgene integration. Several muscle groups in line Tg1888 displayed the presence of the minidystrophin protein as assessed by immunoblot. Expression of the transgene in the skeletal muscles did not exceed that of the endogenous full-length dystrophin protein. The mini-dystrophin protein was localized to the sarcolemma of skeletal muscles from transgenic mdx mice and this was sufficient to restore the DAG complex as well. Finally, we showed that specific muscles from transgenic mdx mice displayed normal morphological characteristics, had restored force in contractility measurements, and were protected from exercise-induced damage. Thus, we conclude that the mouse dystrophin muscle promoter/enhancer used in this study represents an alternative to the MCK and a-actin promoters for use in gene therapeutic approaches for the treatment of DMD. Detailed analysis of the tissue distribution of the transgene product in Tg1888 mice revealed that most of the skeletal muscles tested displayed detectable protein by immunoblot (Fig. 1). Although the transgene was not expressed in tissues like liver and kidney, it was expressed in skeletal muscle and brain, suggesting that the muscle promoter/enhancer sequence was capable of activity in these tissues. This suggests that the muscle promoter/ enhancer retains specificity to dystrophin-expressing tissues. Expression in the heart was less obvious although low levels of transgene transcripts were detected. Indeed, we have previously shown that the mouse dystrophin

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enhancer from intron 1 can target the expression of a lacZ reporter gene to cardiac muscle of transgenic mice [32]. Thus, the variability seen here may reflect differences in sites of integration. Analysis of the TA muscle from transgenic mdx mice revealed that the mini-dystrophin protein was distributed at the sarcolemma of all muscle fibers. However, the level of sarcolemmal dystrophin in mdx/tg muscle was reduced compared with wild-type muscle. Approximately 27% of the fibers in the TA muscle were deemed to be totally strongly positive for dystrophin staining at the sarcolemma. The level of expression was sufficient to ensure an even distribution of fiber size diameter in the muscle of transgenic mdx mice. These fibers were thus healthier appearing and had fewer centralized nuclei than did those from the mdx muscle. The decrease in centrally nucleated muscle fibers is a strong indication of the success of a therapeutic intervention in a muscle wasting disease. Nevertheless, it is of importance to demonstrate that this pathological rescue is accompanied by a further protection of the muscle from mechanically induced stress. Indeed this is an essential consideration for mdx mice, which exhibit degeneration and necrosis of skeletal muscle beginning a few weeks after birth. However, this muscle dystrophy is not sufficient to affect functional properties grossly and the mice have normal motor activity. It is only after exposure to an eccentric exercise regimen that the muscle fibers of mdx mice become more vulnerable to damage [33]. Compared to normal mice, mdx mice show an increase in Evans blue dye penetration into their muscle fibers after a period of downhill running on a treadmill [33]. Here, we have demonstrated that the transgenic mdx mice are better able to resist the exercise-induced muscle damage than are mdx mice (Table 2). Several different muscles were assessed from at least two mice per group. Although most of the muscle groups from transgenic mdx mice displayed a reduction in the total area of damage as visualized by Evans blue dye penetration, the diaphragm was an exception. This respiratory muscle had significant fiber damage even in sedentary mice, and this damage was further exasperated after the exercise protocol. Immunoblot analysis of protein from diaphragm muscle of transgenic mdx mice revealed that the level of mini-dystrophin expression was low, especially compared to the TA muscle (Fig. 2H). Furthermore, our immunofluorescence studies demonstrated that the expression of mini-dystrophin was not equal in all fibers of the diaphragm, and it appeared that some fibers had no expression at all (Figs. 2E–2G). This result suggests that the mosaic expression of the transgene may be caused by a position effect at the site of integration and implies that variable expression of the transgene may not be sufficient for total rescue of this muscle. Another observation from our studies was the increased level of mini-dystrophin with the transgene

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in the mdx background. When TA and diaphragm muscle extracts from transgenic mice on the B6C3F1 background were compared to those from the transgenic mdx mice, it was clear from the immunoblots that the levels of minidystrophin were higher in the latter case (Figs. 1D and 2H). A likely explanation for this phenomenon is that mini-dystrophin accumulates on the membrane to higher levels in mdx than in wild-type mice since it does not have to compete for binding sites with endogenous, full-length dystrophin. The studies described in the present report provide significant information on the functionality of the mouse dystrophin muscle promoter/enhancer sequence to provide expression of associated transgenes within skeletal muscle. Our transgenic mouse studies demonstrate that although this promoter/enhancer can be prone to position effect of transgene integration, we are still able to attain a functional rescue of most of the skeletal muscles in mdx mice. Thus, incorporation of this regulatory cassette into gene therapy vectors being developed for the treatment of DMD may be a viable option.

MATERIALS

AND

METHODS

The mouse dystrophin muscle promoter/enhancer-dystrophin minigene construct. The transgene construct consisted of the mouse dystrophin muscle promoter/enhancer driving the expression of the human dystrophin minigene. The mouse dystrophin muscle enhancer is contained within a 3-kb fragment that has been positioned upstream of the first 850 bp of the mouse dystrophin muscle promoter (Fig. 1A). This dystrophin muscle promoter/enhancer element has been described previously [31,32]. Downstream of these two elements, we have cloned the human dystrophin minigene that lacks exons 17–48 and that measures approximately 6.7 kb [11]. Finally, an SV40 polyadenylation signal has been placed at the 3V end of this fusion gene. The resulting 11.3-kb fragment was used in the generation of transgenic mice. Transgenic mice. Care and use of experimental mice followed the guidelines established by the Canadian Council on Animal Care. To generate transgenic mice, hybrid C57BL/6–C3H F1 mice (produced by crossing C57BL/6 female mice with C3H male mice; obtained from Charles River) were used as donors for fertilized one-cell embryos. Pronuclear microinjection of the transgene construct was performed at a concentration of 3 ng/Al. Zygotes were cultured overnight at 378C in M16 medium under oil. The following day, two-cell-stage embryos were subjected to oviduct transfers in pseudopregnant female CD-1 mice. Tail biopsies were obtained from potential founder mice, DNA was extracted, and transgenic mice were identified by PCR amplification. Breeding of the transgene onto the mdx background was established by crossing the mini-dystrophin transgenic mice (heretofore referred to as tg mice) with mdx female mice (obtained from The Jackson Laboratory). Transgenic male offspring with the mdx allele were identified and used in subsequent experiments. Nontransgenic littermates served as controls. RT-PCR analysis. For RT-PCR analysis, RNA was isolated from different tissues of 2-month-old mice. For cDNA production, equal amounts of total RNA were reverse-transcribed in a standard reaction with MuLV reverse transcriptase (Invitrogen). PCR was performed using 35 cycles in a thermocycler. Amplification of the 440-bp product was performed using the 20-mer Forward Primer 5V-TGCCTTTTTAGTGCATGGCT-3V (specific to the beginning sequence of dystrophin exon 15) and the 20-mer Reverse Primer 5V-AGTAAACGGTTTACCGCCTT-3V (specific to the middle of

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dystrophin exon 50). Actin primers were used as positive controls. The PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide. Amplicons were visualized by UV transillumination. Histological analysis. Tibialis anterior skeletal muscles were collected from wild-type, transgenic, mdx, and mdx/transgenic mice at 4 weeks. Muscles were dissected in PBS, embedded in OCT compound (Sakura), and frozen in liquid nitrogen. Cryostat sections of 10-Am thickness were stored at 208C before use. Sections were then stained with hematoxylin and eosin and examined by light microscopy using a Zeiss Axioplan microscope. Photographed images were imported into the AxioVision 4.1 software with which measurements of the cross-sectional area of each muscle fiber were determined. Extract preparation and immunoblotting. Tibialis anterior muscles, hearts, and diaphragms from at least four wild-type, transgenic, mdx, and mdx/transgenic mice were minced in RIPA lysis buffer (50 mM Tris– HCl, 150 mM NaCl, 0.1% SDS, 0.5% Na deoxycholate, 1.0% Triton X-100) containing protease inhibitors (1 mM PMSF, 0.01 mg/ml aprotinin, 0.01 mg/ml pepstatin, 0.01 mg/ml leupeptin, and 10 mM Na2VO4). Lysates from TA and diaphragm muscle were centrifuged at 6000 rpm and lysates from hearts at 13,000 rpm, each for 5 min. The protein content of the supernatant was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories). Each sample (50 Ag per lane) was electrophoresed on an SDS–polyacrylamide gel (5% stacking, 6% resolving) for 2 h at 100 V and then electrotransferred for 4 h, 0.3 A, at 48C. Membranes were blocked using 5% nonfat dry milk in TBST (100 mM Tris–HCl, pH 8.0, 167 mM NaCl, 0.1% Tween 20) for 1 h and incubated with a 1:20 dilution of the primary antibody in blocking buffer for 1 h at room temperature, washed, and probed with horseradish peroxidase-conjugated goat anti-mouse antisera (Bio-Rad Laboratories) at a 1:750 dilution. Blots were developed using the ECL Plus chemiluminescence system (Amersham Biosciences). Immunohistochemistry. TA skeletal muscles (3 weeks) and diaphragms (7 weeks) were collected from at least three wild-type, transgenic, mdx, and mdx/transgenic mice. Samples were dissected in PBS, embedded in OCT compound (Sakura), and frozen in liquid nitrogen. Cryostat sections of 10-Am thickness were stored at 208C before use. Protocols from the MOM Kit (Vector Laboratories, Inc.) were followed for detecting mouse primary antibodies on mouse tissue with fluorescein. Slides were mounted with antifade reagent in glycerol buffer (Slowfade Light Antifade Kit; Molecular Probes) and analyzed by fluorescence microscopy using a Zeiss Axioplan microscope. Mouse monoclonal antibodies against h-dystroglycan, g-sarcoglycan, and dystrophin (mAb NCL-DYS2) were obtained from Novacastra Labs. Treadmill running. Seven-week-old B6C3F1 male and female, mdx male, and mdx/tg male mice were used for the experiment. Mice were placed on a treadmill with a downward incline of 158. The mice were run for 10 min at a speed of 10 m/min on 3 consecutive days. After the running on day 2, both exercised and sedentary mice were injected intraperitoneally with Evans blue dye (1 mg dye/0.1 ml/10 g body wt), which was prepared by dissolving dye in PBS and sterilizing by filtration through membrane with pore size of 0.2 Am. Mice were dissected 24 h postinjection after the third and final run. BB, EDL, TA, G, SOL, and DIA were dissected in PBS and frozen in OCT. Cryostat sections of 30-Am thickness were made and stored at 208C before use. Sections were rinsed in PBS followed by water and mounted with antifade reagent in glycerol buffer (Slowfade Light Antifade Kit; Molecular Probes). Photographs were taken on a Zeiss microscope and imported into the AxioVision 4.1 software with which the damaged and total fiber areas were measured. The area of Evans blue staining over the total area of the muscle section gave the percentage of the damaged area. Force measurements. Mouse soleus tendons from five wild-type, five mdx, and five mdx/tg mice were tied with surgical silk (6-O) and were constantly immersed in physiological saline solution containing 118.5 mM NaCl, 4.7 mM KCl, 2.4 mM CaCl2, 3.1 mM MgCl2, 25 mM NaHCO3, 2 mM NaH2PO4, and 5.5 mM d-glucose. All solutions were continuously

MOLECULAR THERAPY Vol. 14, No. 5, November 2006 Copyright C The American Society of Gene Therapy

bubbled with 95% O2: 5% CO2 and had a pH 7.4. Experiments were performed at the physiological temperature of 378C. Muscle length was adjusted to get maximum tetanic force and was allowed 30 min equilibrium prior to any measurements. Throughout the experiment, one twitch or one tetanic contraction was elicited every 100 s; tetanic stimulations consisted of a 200-ms train of 0.3-ms, 10-V (supramaximal voltage) pulses at frequencies between 10 and 200 Hz. The stimulating current, which passes between parallel platinum wires located on opposite sides of the muscle, was generated with a Grass S88 stimulator and Grass SIU5 isolation unit (Grass, West Warwick, RI). Force was measured with a Kulite semiconductor strain gauge (Model BG100, Vancouver, BC, Canada) and digitized at 5 kHz with a Keithley Metrabyte A-D board (Model DAS50; Edmonton, AB, Canada). Peak force, half-rise time, half-relaxation time, width, maximum rate of force development, and relaxation were analyzed on a computer as described previously [34]. Statistical analysis. For the contractile properties, t tests were used to determine significant differences between control, mdx, and mdx/tg mice. For the force–frequency curve, split-plot ANOVA designs were used to test for significance with the treatment bmouseQ in the whole plot because muscles were from different mice and the treatment bfrequencyQ in the split plot because peak force at different frequencies was obtained from the same muscles. ANOVA calculations were made using the General Linear Model procedures of the Statistical Analysis Software (SAS Institute Inc., Cary, NC, USA). When a main effect or an interaction was significant, the least-square difference (LSD) was used to locate the significant differences.

ACKNOWLEDGMENTS We are grateful to Robin Parks for critical reading of the manuscript and the rest of the Kothary laboratory for helpful discussions. Thanks to the JesseTs Journey Foundation for Gene and Cell Therapy for their generous support of our research program. This project was funded by grants from the Canadian Institutes of Health Research and the Muscular Dystrophy Association (USA) to R.K., from the National Science and Engineering Research Council to J-M.R., and from the Canadian Genetic Diseases Network and the Heart and Stroke Foundation of Ontario to R.G.W. RECEIVED FOR PUBLICATION JUNE 8, 2005; REVISED MARCH 15, 2006; ACCEPTED APRIL 16, 2006.

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