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 Kothary 1,2,3,5, * 1 Ottawa Health Research Institute, Ottawa, ON, Canada K1H 8L6 2 Center for Neuromuscular Disease, 3 Department of Cellular and Molecular Medicine, 4 Department of Biochemistry, Microbiology, and Immunology, and 5 Department of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 *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 dis- eases that are caused by defective expression of dystro- phin [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 develop- ment of such strategies a daunting challenge (reviewed in [7]). However, work by Chamberlain and others has shown that expression of dystrophin mini- and micro- genes may be a viable option for the treatment of DMD [8–17]. Identification of promoters that ensure appropri- ate 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 regu- latory 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 regu- latory 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- ARTICLE doi:10.1016/j.ymthe.2006.04.013 MOLECULAR THERAPY Vol. 14, No. 5, November 2006 724 Copyright C The American Society of Gene Therapy 1525-0016/$30.00
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ARTICLE doi:10.1016/j.ymthe.2006.04.013
The Mouse Dystrophin Muscle Promoter/EnhancerDrives Expression of Mini-dystrophin in Transgenicmdx 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,*
1Ottawa Health Research Institute, Ottawa, ON, Canada K1H 8L62Center 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
*To whom correspondence and reprint requests should be addressed at the Ottawa Health Research Institute, 501 Smyth Road,
Successful gene therapy for Duchenne muscular dystrophy (DMD) requires the restoration ofdystrophin protein in skeletal muscles. To achieve this goal, appropriate regulatory elements thatimpart tissue-specific transgene expression need to be identified. Currently, most muscle-directedgene therapy studies utilize the muscle creatine kinase promoter. We have previously described amuscle enhancer element (mDME-1) derived from the mouse dystrophin gene that increasestranscription from the mouse dystrophin muscle promoter. Here, we explore the use of this nativemouse dystrophin muscle promoter/enhancer to drive expression of a human dystrophin minigenein transgenic mice. We show that the dystrophin promoter can provide tissue-specific transgeneexpression and that the mini-dystrophin protein is expressed at the sarcolemma of skeletal musclesfrom mdx mice, where it restores the dystrophin-associated glycoprotein complex. The level oftransgene expression obtained is sufficient to protect mdx muscles from the morphological andphysiological symptoms of muscular dystrophy, as well as from exercise-induced damage. Therefore,the dystrophin muscle promoter/enhancer sequence represents an alternative for use in genetherapy vectors for the treatment of DMD.
Duchenne and Becker muscular dystrophies (DMD/BMD)are X-chromosome-linked recessive muscle wasting dis-eases that are caused by defective expression of dystro-phin [1,2]. The dystrophin gene spans 2.9 Mb. It consistsof 79 exons and is expressed as a 14-kb transcript inmuscle cells [3,4]. A mouse model for DMD is the mdxmouse [5], which features a point mutation in exon 23 ofthe dystrophin gene [6]. Although the mdx mouse doesnot display any overt signs of muscle weakness ormovement difficulty, its phenotype is characterized bycycles of muscle degeneration/regeneration.
Replacement of dystrophin by gene therapy is onestrategy to slow the progression of DMD but the large sizeof the gene and corresponding mRNA makes develop-ment of such strategies a daunting challenge (reviewed in[7]). However, work by Chamberlain and others has
shown that expression of dystrophin mini- and micro-genes may be a viable option for the treatment of DMD[8–17]. Identification of promoters that ensure appropri-ate tissue-specific expression of the therapeutic gene isthus important for achieving gene therapy for DMD.
Previous efforts have utilized dystrophin mini- andmicrogenes under the control of the muscle creatinekinase (MCK) gene promoter/enhancer to achievemuscle-specific expression [8,10,15,18]. The MCK regu-latory elements have been well characterized, both incell culture and in transgenic mice [8,19–21]. Initiallycharacterized as a 6.5-kb promoter/enhancer segment,smaller versions also function as muscle-specific regu-latory elements, including for example a 1.35-kb MCKpromoter/enhancer element driving the expression of amini-dystrophin gene in transgenic mice [8]. With thispromoter, the muscular dystrophy phenotype was atte-
nuated in fast, but not slow, muscles of transgenic mdxmice. However, this latter study was with a single line oftransgenic mice and therefore the fiber-type specificitymay have been due to the site of transgene integration.Overall, although the MCK promoter has shown greatpromise in its use as a muscle-specific regulatoryelement, the dystrophin gene promoter also deservesconsideration.
To date there have been eight promoters identified atthe DMD locus producing eight different tissue-specificisoforms (reviewed in [22]). Of these, only the muscle,brain, and cerebellar Purkinje promoters produce tran-scripts encoding a full-length 427-kDa dystrophin protein[23–27]. The first 150 nucleotides of the muscle promoterare required for muscle-specific expression in culturedcells [28]. A transcriptional enhancer (dystrophin muscleenhancer-1) has been identified 6.5 kb downstream of themuscle promoter within muscle intron 1 of the humandystrophin gene [29]. This enhancer increases transcrip-tion from the dystrophin muscle promoter in bothmyoblasts and myotubes [30]. Likewise, we characterizedan intron-1 enhancer element (mouse dystrophin muscleenhancer-1; mDME-1) 8.5 kb downstream of the mousedystrophin muscle promoter [31]. A 3-kb fragmentharboring mDME-1 increased transcription from themouse dystrophin muscle promoter in cultured myo-tubes. In transgenic mice, a mouse dystrophin musclepromoter/enhancer–lacZ transgene was expressed in bothskeletal muscle and compartments of the heart [32].Thus, the dystrophin muscle promoter is dependent onthe enhancer sequence to target both skeletal and heartmuscle.
To determine whether the mouse dystrophin musclepromoter/enhancer has any therapeutic potential, wehave generated transgenic mice expressing the humandystrophin minigene. We demonstrate that this pro-moter/enhancer combination is capable of impartingtransgene expression to skeletal muscle and that this issufficient to restore the wild-type phenotype in mdxmice. Muscles from the transgenic mdx mice are pro-tected against exercise-induced damage and are notmorphologically or physiologically different from theirwild-type littermates. This work highlights the potentialof using dystrophinTs own regulatory elements in genetherapy for DMD.
RESULTS
Generation of Dystrophin Muscle Promoter/EnhancerDystrophin Minigene Transgenic MiceWe have used our previously characterized mouse dys-trophin muscle promoter/enhancer cassette [31,32] todrive muscle-specific expression of the human dystro-phin minigene (Fig. 1A) in transgenic mice. We obtainedfour founder mice and our initial analysis was directed atexamining 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-tran-scriptase polymerase chain reaction (RT-PCR) that thetransgene is expressed in several different muscle types inline Tg1888 (Fig. 1B, top). Interestingly, the transgene isalso expressed in brain and at a reduced level in the heartof these mice. By contrast, it is not expressed in the liverand kidney (Fig. 1B). The expression of the transgene inthe second line, Tg1867, was considerably restricted andat a reduced level (Fig. 1B, bottom). This variability intransgene expression likely reflects the influence of site ofintegration.
Tissue-Specific Expression of the Dystrophin MinigenePrevious work has shown that the expression of the full-length 427-kDa dystrophin protein in normal wild-typemice is restricted to skeletal muscle, heart, and brain. Weperformed an immunoblot analysis of protein extractsfrom various tissues of wild-type or Tg1888 mice andhave confirmed these observations (Fig. 1C). The full-length dystrophin protein is detected in several musclegroups and in heart. It is also present in brain at reducedlevels. As expected, the endogenous dystrophin proteinis not detected in liver and kidney (Fig. 1C). Weperformed further immunoblot assays and, consistentwith the RT-PCR analysis, the transgene product ispresent in several muscle groups of line Tg1888 (Fig.1C). The mini-dystrophin protein is also detected inbrain, but not in liver and kidney. We were unable todetect transgene-derived mini-dystrophin in tissues fromthe second line, Tg1867 (data not shown). We usedstrain Tg1888 for all subsequent experiments presentedin this paper. The transgene was bred onto the mdxbackground by crossing Tg1888 male mice with mdxfemales. Male offspring were screened for the presence ofthe transgene to distinguish between mdx and mdx/tgmice.
Analysis of protein extracts from tibialis anterior (TA)muscle reveals the presence of the full-length dystrophinprotein in the wild-type mice and in the Tg1888 mice,but not in the mdx and mdx/tg mice (Fig. 1D). Asexpected, the mini-dystrophin protein is expressed onlyin the Tg1888 and mdx/tg mice (Fig. 1D), and the level ofexpression of mini-dystrophin is substantially greater inthe mdx background, a consistent observation in thisstudy (e.g., see Fig. 2H).
Immunofluorescence analysis of the TA muscles fromwild-type, mdx, and mdx/tg mice revealed the expectedsarcolemmal distribution of full-length dystrophin in thewild-type muscle (Fig. 2A) but its absence in the mdxmuscle (Fig. 2B). Parallel analysis of TA muscle sectionsfrom mdx/tg mice demonstrates sarcolemmal localizationof the mini-dystrophin protein (Fig. 2C). The level ofsarcolemmal mini-dystrophin in mdx/tg muscle waslower compared with sarcolemmal dystrophin in wild-type muscle. However, from our examination of several
FIG. 1. Derivation of transgenic mice and expression analysis. (A) Schematic representations of dystrophin and related constructs. Structural domains of full-
length dystrophin, mini-dystrophin, and the mouse dystrophin muscle promoter/muscle enhancer mini-dystrophin transgene construct are shown. The full-
length 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),
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. Full-
length 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.
ARTICLE doi:10.1016/j.ymthe.2006.04.013
samples, virtually every muscle fiber of the mdx/tgbackground was dystrophin positive. We counted thenumber of total strongly dystrophin positive muscle
726
fibers in four different mdx/tg mice. We made theassumption that btotal strongly positiveQ fibers shouldrequire that at least 95% of the perimeter of the fiber be
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 full-
length 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.
ARTICLEdoi:10.1016/j.ymthe.2006.04.013
very bright. We measured the percentage of total stronglypositive fibers and the numbers were consistent in allfour mice (27, 26.6, 27.6, and 27.5%).
We next examined the diaphragm of the mdx/tg micefor expression of the mini-dystrophin protein. Staining ofcontrol diaphragm muscle sections demonstrates normal
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sarcolemmal localization of dystrophin, whereas stainingof the mdx diaphragm muscle reveals the absence ofdystrophin (Figs. 2E and 2F). Parallel analysis of dia-phragm muscle sections from mdx/tg mice demonstratesthat the minigene is expressed and that the mini-dystrophin protein is localized at the sarcolemma (Fig.
2G). Curiously, not all fibers within the diaphragmexpress mini-dystrophin at the sarcolemma. Further-more, immunoblot analysis of proteins extracted fromthe diaphragm muscle reveals that although the mini-dystrophin protein is detectable in mdx/tg mice, it is at alevel much lower than that in the TA muscle of the samemouse (Fig. 2H).
Restoration of the DAG Complex in the mdx/tg MiceAblation of dystrophin or members of the dystrophin-associated glycoprotein (DAG) complex generally resultsin a down-regulation of other members of this complex.We performed a study on how the distribution of the DAGcomplex is affected in mdx and mdx/tg skeletal muscle. Wesectioned TA muscles from 3-week-old mice and immu-nostained them with antisera against dystrophin, h-dystroglycan, and g-sarcoglycan (Fig. 3). As expected, theDAG complex protein distribution is normal in wild-typemuscle and localization to the sarcolemma is observed(Figs. 3A, 3D, and 3G). In contrast, dystrophin, as well as h-
FIG. 3. Restoration of the dystrophin-associated glycoprotein (DAG) complex in
wild-type, (B, E, and H) mdx, and (C, F, and I) mdx/tg mice were collected an
complex: dystrophin (DYS), h-dystroglycan (h-DG), and g-sarcoglycan (GSG). Sta
sarcolemma (B, E, and H), whereas control TA muscle sections demonstrate norm
TA muscle sections from mdx/tg mice demonstrates restoration of the DAG com
728
dystroglycan and g-sarcoglycan, is either absent orseverely diminished in the sarcolemma of mdx muscle(Figs. 3B, 3E, and 3H). The loss of the DAG complex fromthe sarcolemma of mdx muscle is corrected by theexpression of the mini-dystrophin protein in the mdx/tgmuscle (Figs. 3C, 3F, and 3I). Thus, the level of expressionof the minigene from the dystrophin muscle promoter/enhancer, although low, is sufficient to ensure restorationof the DAG complex.
Histopathological Analysis of Skeletal MuscleWe performed histopathology of hind-limb skeletalmuscle from 4-week-old mice to determine whetherexpression of mini-dystrophin protein from the dystro-phin muscle promoter/enhancer was sufficient to preventpathology. Hematoxylin–eosin stained cross sections ofwild-type and transgenic TA muscle have normal mor-phology, with consistency in fiber caliber and minimalevidence of central nuclei (Figs. 4A and 4B). In contrast,staining of mdx TA muscle sections demonstrates mor-
mdx/tg mice. Tibialis anterior muscles from 3-week-old (A, D, and G) B6C3F1
d sections stained with antisera against the following members of the DAG
ining of mdx TA muscle reveals the absence of DAG complex proteins from the
al sarcolemmal localization of these proteins (A, D, and G). Parallel analysis o
plex proteins to the sarcolemma (C, F, and I). Scale bar, 50 Am.
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.
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phological characteristics of dystrophy, including varia-tion in fiber size, mononuclear cell infiltrates, fibrosis,and abundant centrally located myonuclei (Fig. 4C).Conversely, parallel analysis of TA muscle sections frommdx/tg mice demonstrates a broader range of fiber sizewith fewer central nuclei and healthier looking fibers (Fig.4D). Quantification of fiber cross-sectional areas revealedthat the fibers (n = 300 fibers) in sections of muscle fromwild-type or transgenic mice have a normal distribution
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pattern (Fig. 4E). Similarly, we determined the cross-sectional area of individual fibers for mdx (n = 300 fibers)and mdx/tg (n = 268 fibers) muscle, and the resultsindicated that there are greater numbers of small-caliberfibers in the mdx muscle, whereas the mdx/tg muscleshave more large fibers (Fig. 4F).
We also quantified the occurrence of centrallynucleated fibers in the same TA muscle sections used forthe histopathology. The muscles from wild-type and
TABLE 2: Distribution of damaged areas (in %) in differenex
Muscle Sedentary wt Exercised wt Sedentary mdx
TA 0 0 0 0 0 0.30
EDL 0 0 0 0 4.60 0SOL 0 0 0 0.18 0 5.46
G 0 0 0 0 3.24 9.21
BB 0 0 0 0 0.78 1.25
DIA 0 0 0 0 3.43 6.00
Values from two mice per category are shown in each column. n.d., not determined.
ARTICLE doi:10.1016/j.ymthe.2006.04.013
730
TABLE 1: Quantification of centrally located nuclei in TAmuscle fibers of control, mdx, and mdx/tg mice
Genotype Fibers with centrallylocated nucleus
% Central nucle
wt 3 of 300 1
tg 2 of 300 0.7
mdx 92 of 300 30.7mdx/tg 23 of 268 8.6
i
B6C3F1/tg mice have less than 1% centrally nucleatedmyofibers, whereas those from mdx mice have 30.7%centrally nucleated fibers (Table 1). Expression of thetransgene in the mdx background decreased the incidenceof centrally nucleated fibers to 8.6% (Table 1). Thus, theexpression of the transgene from the dystrophin musclepromoter/enhancer is capable of at least partially rescuingthe dystrophic phenotype in mdx muscles.
Protection From Exercise-Induced Muscle DamageWe next determined whether dystrophin muscle pro-moter/enhancer-driven mini-dystrophin expression iscapable of providing protection to exercise-inducedmuscle damage. We put the mice either in abnonexercisedQ sedentary group or in an exercised group,which was subjected to a regimen of running on atreadmill for 10 min/day for a period of 3 days [33]. Wethen visualized degenerating muscle fibers by in vivostaining with Evans blue dye. We analyzed several muscletypes, and to quantify the observations better, wemeasured the area of muscle cross section labeled withthe Evans blue dye and expressed it as a percentage of thetotal area. We did this for several muscles, including theTA, extensor digitorum longus (EDL), soleus (SOL), andgastrocnemius (G), which together represent the hind-limb muscles. As well, we performed analysis of thebiceps brachii (BB) as representatives of the forelimbmuscles. Finally, we included the diaphragm (DIA) as arespiratory muscle. The results obtained from two mice ofeach group are summarized in Table 2. The only damagedfibers observed in the sedentary or exercised wild-typemice were a small cluster in the soleus of one exercisedwild-type mouse. In contrast, several muscles from thesedentary mdx mice displayed significant muscle damage.
t merc
After 3 days of exercise, there was a general accentuationof muscle fiber damage in some of the muscles in the mdxmice, with the most notable effect being in the TA, theBB, and the diaphragm muscles. The expression of mini-dystrophin reduced the occurrence of damage in the TA,EDL, SOL, G, and BB muscles of mdx/tg mice. Indeed, theamount of muscle damage in the hind-limb and forelimbmuscles of sedentary mdx/tg mice approached thatobserved for the wild-type mice. Furthermore, the trans-gene appeared to provide sufficient protection fromexercise-induced muscle damage for mdx/tg mice. Theone exception to this was in the diaphragm, where thedamage already present in sedentary mdx/tg mice wasworsened after exercise, as it was in the diaphragm frommdx mice. This is consistent with our observation ofmosaic expression of the transgene in the diaphragm (Fig.2G). Taken together, our results suggest that the dystro-phin muscle promoter/enhancer can drive sufficientexpression of the mini-dystrophin gene to most skeletalmuscle types to impart protection from exercise-induceddamage.
Mini-dystrophin Expression Restores MuscleContractility in Soleus Muscle of mdx MiceTo determine whether the mini-dystrophin expression inour mdx/tg mice could improve muscle contractility, wechose 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 measurementsas described under Materials and Methods. We firstexamined the contractile properties during twitch andtetanic contraction at the physiological temperature of378C. Most of the twitch parameters were not signifi-cantly different between wild type, mdx, and mdx/tgmice (Table 3). The mean peak tetanic force wassignificantly less in mdx soleus compared to wild-typesoleus, while the maximum rates of force developmentand relaxation were lower but not significantly different.All three parameters were significantly greater in mdx/tgsoleus than in mdx soleus. From the force–frequencyrelationship between the various samples, we alsoobserved that the peak forces of mdx soleus weresignificantly less than those from wild-type soleus whenthe stimulation frequencies were 120 Hz or greater (Fig.
uscles of control, mdx, and mdx/tg mice and the effects ofise
TABLE 3: Contractile properties of control, mdx, and mdx/tg soleus muscle at 378C
Parameter wt mdx mdx/tg
Twitch
Peak force (N/cm2) 2.3 F 0.5 2.8 F 0.4 2.3 F 0.7
Half-rise time (ms) 2.7 F 0.1 2.9 F 0.2 3.0 F 0.1
Time to peak (ms) 9.4 F 0.5 11.4 F 0.2* 10.6 F 0.5Half-relaxation time (ms) 10.4 F 0.4 11.8 F 1.4 9.6 F 0.9
Width (ms) 17.2 F 0.7 20.3 F 1.6 17.2 F 1.1
Maximum rate of force development (N/cm2s) 378 F 92 419 F 50 364 F 98
Maximum rate of relaxation (N/cm2s) 155 F 33 164 F 27 158 F 30
Tetanus (200 Hz)
Peak force (N/cm2) 32.3 F 4.9 23.9 F 2.3* 32.4 F 2.5**Half-rise time (ms) 20.0 F 1.1 16.3 F 0.5* 16.0 F 0.5*
Half-relaxation time (ms) 45.9 F 0.7 45.7 F 2.9 40.4 F 1.6*
Maximum rate of force development (N/cm2s) 954 F 202 810 F 65 1132 F 87**
Maximum rate of relaxation (N/cm2s) 1203 F 198 860 F 108 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.
ARTICLEdoi:10.1016/j.ymthe.2006.04.013
5). However, the peak forces of mdx/tg soleus were notonly significantly greater than those of mdx soleus, theywere also similar to the peak forces of wild-type soleus(Fig. 5).
DISCUSSION
In the present study, we have used the native dystro-phin 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).
MOLECULAR THERAPY Vol. 14, No. 5, November 2006
Copyright C The American Society of Gene Therapy
dystrophin minigene in transgenic mice. Analysis oftwo different transgenic lines revealed that this pro-moter/enhancer sequence has the capability to drivetransgene expression to muscle tissues. However, therewas variability in expression, and line Tg1888 displayeda broader range of tissue expression than did lineTg1867. This difference is likely due to an influence ofthe site of transgene integration. Several muscle groupsin line Tg1888 displayed the presence of the mini-dystrophin protein as assessed by immunoblot. Expres-sion of the transgene in the skeletal muscles did notexceed that of the endogenous full-length dystrophinprotein. The mini-dystrophin protein was localized tothe sarcolemma of skeletal muscles from transgenic mdxmice and this was sufficient to restore the DAG complexas well. Finally, we showed that specific muscles fromtransgenic mdx mice displayed normal morphologicalcharacteristics, had restored force in contractility meas-urements, and were protected from exercise-induceddamage. Thus, we conclude that the mouse dystrophinmuscle promoter/enhancer used in this study representsan alternative to the MCK and a-actin promoters for usein gene therapeutic approaches for the treatment ofDMD.
Detailed analysis of the tissue distribution of thetransgene product in Tg1888 mice revealed that most ofthe skeletal muscles tested displayed detectable proteinby immunoblot (Fig. 1). Although the transgene was notexpressed in tissues like liver and kidney, it was expressedin skeletal muscle and brain, suggesting that the musclepromoter/enhancer sequence was capable of activity inthese tissues. This suggests that the muscle promoter/enhancer retains specificity to dystrophin-expressingtissues. Expression in the heart was less obvious althoughlow levels of transgene transcripts were detected. Indeed,we have previously shown that the mouse dystrophin
enhancer from intron 1 can target the expression of alacZ reporter gene to cardiac muscle of transgenic mice[32]. Thus, the variability seen here may reflect differ-ences in sites of integration.
Analysis of the TA muscle from transgenic mdx micerevealed that the mini-dystrophin protein was distributedat the sarcolemma of all muscle fibers. However, the levelof sarcolemmal dystrophin in mdx/tg muscle was reducedcompared with wild-type muscle. Approximately 27% ofthe fibers in the TA muscle were deemed to be totallystrongly positive for dystrophin staining at the sarco-lemma. The level of expression was sufficient to ensurean even distribution of fiber size diameter in the muscleof transgenic mdx mice. These fibers were thus healthierappearing and had fewer centralized nuclei than didthose from the mdx muscle.
The decrease in centrally nucleated muscle fibers is astrong indication of the success of a therapeutic inter-vention in a muscle wasting disease. Nevertheless, it is ofimportance to demonstrate that this pathological rescueis accompanied by a further protection of the musclefrom mechanically induced stress. Indeed this is anessential consideration for mdx mice, which exhibitdegeneration and necrosis of skeletal muscle beginninga few weeks after birth. However, this muscle dystrophy isnot sufficient to affect functional properties grossly andthe mice have normal motor activity. It is only afterexposure to an eccentric exercise regimen that the musclefibers of mdx mice become more vulnerable to damage[33]. Compared to normal mice, mdx mice show anincrease in Evans blue dye penetration into their musclefibers after a period of downhill running on a treadmill[33]. Here, we have demonstrated that the transgenicmdx mice are better able to resist the exercise-inducedmuscle damage than are mdx mice (Table 2). Severaldifferent muscles were assessed from at least two mice pergroup. Although most of the muscle groups from trans-genic mdx mice displayed a reduction in the total area ofdamage as visualized by Evans blue dye penetration, thediaphragm was an exception. This respiratory muscle hadsignificant fiber damage even in sedentary mice, and thisdamage was further exasperated after the exercise proto-col. Immunoblot analysis of protein from diaphragmmuscle of transgenic mdx mice revealed that the level ofmini-dystrophin expression was low, especially comparedto the TA muscle (Fig. 2H). Furthermore, our immuno-fluorescence studies demonstrated that the expression ofmini-dystrophin was not equal in all fibers of thediaphragm, and it appeared that some fibers had noexpression at all (Figs. 2E–2G). This result suggests thatthe mosaic expression of the transgene may be caused bya position effect at the site of integration and implies thatvariable expression of the transgene may not be sufficientfor total rescue of this muscle.
Another observation from our studies was theincreased level of mini-dystrophin with the transgene
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in the mdx background. When TA and diaphragm muscleextracts from transgenic mice on the B6C3F1 backgroundwere compared to those from the transgenic mdx mice, itwas clear from the immunoblots that the levels of mini-dystrophin were higher in the latter case (Figs. 1D and2H). A likely explanation for this phenomenon is thatmini-dystrophin accumulates on the membrane tohigher levels in mdx than in wild-type mice since it doesnot have to compete for binding sites with endogenous,full-length dystrophin.
The studies described in the present report providesignificant information on the functionality of themouse dystrophin muscle promoter/enhancer sequenceto provide expression of associated transgenes withinskeletal muscle. Our transgenic mouse studies demon-strate that although this promoter/enhancer can beprone to position effect of transgene integration, weare still able to attain a functional rescue of most of theskeletal muscles in mdx mice. Thus, incorporation of thisregulatory cassette into gene therapy vectors beingdeveloped for the treatment of DMD may be a viableoption.
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 dystro-
phin 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 approx-
imately 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