BBRC Biochemical and Biophysical Research Communications 293 (2002) 1265–1272 www.academicpress.com
Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene Miki Sakamoto,a Katsutoshi Yuasa,a Madoka Yoshimura,a Toshifumi Yokota,a Takaaki Ikemoto,b Misao Suzuki,c George Dickson,d Yuko Miyagoe-Suzuki,a and Shin’ichi Takedaa,* a
c
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa Higashi, Kodaira, Tokyo 187-8502, Japan b Department of Pharmacology, Saitama Medical School, Morohongo 38, Moroyama-machi, Saitama 350-0495, Japan Division of Transgenic Technology, Center for Animal Resources and Development, Kumamoto University, 1-1-1 Honjou, Kumamoto 860-8556, Japan d Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Surrey TW20 0EX, UK Received 11 April 2002
Abstract The adeno-associated virus vector is a good tool for gene transfer into skeletal muscle, but the length of a gene that can be incorporated is limited. To develop a gene therapy for Duchenne muscular dystrophy, we generated a series of rod-truncated microdystrophin cDNAs: M3 (one rod repeat, 3.9 kb), AX11 (three rod repeats, 4.4 kb), and CS1 (four rod repeats, 4.9 kb). These microdystrophins, driven by a CAG promoter, were used to produce transgenic (Tg) mdx mice and all three micro-dystrophins were shown to localize at the sarcolemma together with the expression of dystrophin-associated proteins. Among them, CS1 greatly improved dystrophic phenotypes of mdx mice and contractile force of the diaphragm in particular was restored to the level of normal C57BL/10 mice. AX11 modestly ameliorated the dystrophic pathology, but, importantly, M3-Tg mdx mice still showed severe dystrophic phenotypes. These data suggest that the rod structure, and its length in particular, is crucial for the function of micro-dystrophin. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Dystrophin; Duchenne muscular dystrophy; Gene therapy; mdx Mouse; Mini-dystrophin; Micro-dystrophin; Rod domain; Transgenic mice; Adeno-associated virus vector
Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder of skeletal muscle caused by mutations in the dystrophin gene [1]. Progressive muscle weakness, including the respiratory muscles, cardiomyopathy, and early death, characterizes the disease. There is no effective treatment for the disease at present, although gene therapy could be an attractive approach to the disease. The dystrophin gene measures 2.4 Mb and full-length dystrophin cDNA is about 14 kb [2]. The protein product, dystrophin (427 kDa), is composed of four major structural domains: N-terminal, central rod, cysteine-rich, and C-terminal domains. Dystrophin
*
Corresponding author. Fax: +81-42-346-1750. E-mail address:
[email protected] (S. Takeda).
binds F-actin [3,4] or c-actin filaments via its N-terminal domain [5], whereas the cysteine-rich and C-terminal domains bind integral and peripheral proteins and stabilize the dystrophin-associated protein (DAP) complex at the sarcolemma [6,7]. a-Dystroglycan, one of the DAPs, binds laminin in the basement membrane. Thus, dystrophin forms a strong physical link between the sarcolemma and cytoskeleton [4,5] and provides mechanical reinforcement to the sarcolemma [8]. It is widely accepted that dystrophin deficiency results in disruption of the sarcolemma due to mechanical stress during muscle contraction. In addition, several pieces of evidence suggest that an ischemic process and disruption of cell survival signaling accelerate the degeneration of muscle fibers [9]. Progress toward a therapeutic approach to DMD has been made using several techniques, including gutted
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adenovirus (Ad) vectors, which can carry the full-length dystrophin cDNA. However, clinical use of gutted vectors has been slowed down by production difficulties: low yield and contamination by the helper-type Ad virus [10]. Transduction of adult muscle fibers with an adenoassociated virus (AAV) vector enables efficient and stable expression of the therapeutic genes, but the AAV vector cloning capacity, up to 4.9 kb, is too limited for therapy of DMD. Therefore truncation of dystrophin cDNA to a small size with an almost complete function is required. Among the four domains of dystrophin, the role of the rod domain is not fully understood, but it is speculated to function as a long spacer between the N-terminal actin-binding domain and the cysteine-rich/ C-terminal domain [11]. Surprisingly, it has been shown that a large in-frame deletion in the central rod domain results in Becker muscular dystrophy, an allelic, milder form of DMD [12]. In addition, transgenic (Tg) mdx mice carrying 6.4 kb mini-dystrophin cDNA cloned from a patient exhibited marked amelioration of dystrophic phenotypes [13,14]. This result suggests that a truncated micro-dystrophin with a larger deletion in the central rod domain could also improve dystrophic phenotypes. Based on this hypothesis, we previously constructed three micro-dystrophins and introduced them into mouse skeletal muscles using an Ad vector to examine their function [15]. Even the shortest construct, M3, which had only one rod and two hinges, showed efficient sarcolemmal expression in vivo. The Ad vector, however, is not suitable for the long-term expression of the transferred gene, since expression of the gene product is limited by immune response against the vector [16]. Recently, Wang et al. [17] succeeded in AAV vectormediated gene transfer using micro-dystrophin cDNA approximately 4 kb long and proved its long-term expression, although they did not describe its function in detail. In this study, we produced Tg mdx mice to analyze the functions of micro-dystrophins and to test whether the dystrophic process had been ameliorated or not. We demonstrated that the length of the rod repeats is one of the crucial factors for dystrophin function and that one micro-dystrophin construct with four rod repeats and three hinges (4.9 kb) could completely protect muscle fibers from the dystrophic process.
Materials and methods Construction of human rod-truncated micro-dystrophin cDNAs. We generated a series of rod-truncated micro-dystrophin cDNAs with varying sizes of the rod domain. Micro-dystrophin M3 and AX11 cDNAs were prepared as in our previous report [15] and each cDNA was cloned into the plasmid pCAG containing a CAG expression unit [18], generating pCAGM3 and pCAGAX11, respectively. pCAG was
generated from cosmid pAxCAwt [19] by removing approximately 40 kb of the NruI fragment. CS1 cDNA was newly generated as follows. Two PCRs were independently performed using pBSBMD [15] as the template and primers F1/R1 or F2/R2: F1: GATGAATCTA GTGGAGATCA C; F2: GATTACTGTG GATACCCTTG AAA GACTCCA GGAAC; R1: GTCTTTCAAG GGTATCCACA GTAATCTGCC TCTTC; R2: TTCATGCAGC TGCCTGACTC. These primers correspond to the following sequences of human dystrophin cDNA (GenBank Accession No. M18533): F1: 1760–1780; F2: 2347–2359 and 9002–9023; R1: 9014–9002 and 2359–2338; R2: 9328– 9309. Subsequently, a mixture of two resulting PCR products was used as the template for the second PCR with primer F1/R2. The PCR product was cloned into a pCR2.1 Vector (Invitrogen), generating pCR2.1CS1 and confirmed by DNA sequencing. The AflII/HindIII fragment from pCR 2.1CS1 was inserted into the AflII/HindIII sites of pCAGAX11, generating pCAGCS1. Generation of Tg mdx mice. The DNA fragments containing the expression unit of micro-dystrophins were prepared as follows. pCAGM3, pCAGAX11, and pCAGCS1 were digested with PmeI and then the 6.1, 6.8, and 7.3 kb fragments were purified from lowmelting-point agarose gels using a GELase Agarose Gel-Digesting Preparation Kit (Epicentre Technologies, Madison, WI). Tg mice were generated by one of us (S.M.) for M3-Tg or by Gencom (Machida, Tokyo, Japan) for the other two. Embryos from C57BL/6 mice were micro-injected with the purified DNA fragment. The resulting F0 or F1 male mice were screened by Southern blotting and transgene-positive F0 males were mated with dystrophin-deficient mdx females to obtain Tg F1 mdx male mice. Western blot analysis. Total cellular protein was extracted with a reducing sample buffer (10% SDS, 70 mM Tris–HCl, pH 6.8, 5% b-mercaptoethanol, and 10 mM EDTA) from the tibialis anterior (TA) muscle. The protein concentration was determined by Bio-Rad protein assay. Protein/lane (75 or 25 lg) was separated on a 6% SDS– polyacrylamide gel and electrically transferred to a PVDF membrane (Millipore). After blocking with 3% skim milk, the blot was incubated with an anti-dystrophin monoclonal antibody, NCL-DYS2 (1:30 dilution, Novocastra). After washing with TBST, the blot was incubated with a 1:5000 dilution of HRP-conjugated rabbit anti-mouse IgG1 antibody (Zymed Laboratories). The signal was detected using the enhanced chemiluminescence method (Amersham Biosciences). Histological analysis. Micro-dystrophin-Tg mdx F1 mice were sacrificed when 5- and 10-week-old and the TA muscles and diaphragms were isolated and quickly frozen in liquid nitrogen-cooled isopentane. Normal C57BL/10 (B10) mice were used as positive controls and mdx mice without the transgene in F1 litters as negative controls. Crosssections (10 lm) were stained with hematoxylin and eosin (H&E). Photographs were taken with a microscope, Leica DMRB (Leica), using a digital still camera system HC-2500 (Fuji Film). We counted the number of centrally nucleated fibers and peripherally nucleated fibers in a whole cross-section of B10, mdx, and micro-dystrophin-Tg mdx mice, as previously described by Yamamoto et al. [20]. Immunohistochemistry. Acetone-fixed cryosections (6 lm) were blocked with 5% goat serum and 2% BSA in PBS. Dystrophin was detected by direct immunostaining using the monoclonal antidystrophin antibody MANDRA-1 (Sigma–Aldrich) labeled with the fluorescent dye Alexa 488 (Molecular Probes, Oregon, OR) (1:200 dilution, gift of Dr. M. Imamura, National Institute of Neuroscience, Tokyo, Japan). For detection of DAPs, sections were incubated with the following antibodies: monoclonal antibodies against b-dystroglycan (1:50 dilution) and a-sarcoglycan (1:50 dilution) (NCL-43DAG and NCL-a SARC, Novocastra) and rabbit polyclonal antibody against a1-syntrophin (1:500 dilution) [21]. Primary antibodies were detected by Alexa 488-labeled anti-mouse and anti-rabbit antibodies (Molecular Probes). The signals were recorded by a confocal laser scanning microscope, Leica TCS SP (Leica). Measurement of serum CK. Serum creatine kinase (CK) levels were measured at the SRL Laboratory (Tachikawa, Tokyo, Japan).
M. Sakamoto et al. / Biochemical and Biophysical Research Communications 293 (2002) 1265–1272 Contractile properties of diaphragm. Diaphragm strips 2–2.5 mm wide, including an adjacent section of a single rib and part of the central tendon, were cut from the central region of the lateral costal hemidiaphragm of 5- and 10-week-old mice. A silk suture was tied to each end of the muscle strip [22]. The diaphragm muscle was mounted in a vertical tissue chamber and connected to a force transducer UL-10GR (MINEBEA, Saku, Nagano, Japan) and length servosystem MM-3 (NARISHIGE, Setagaya-ku, Tokyo, Japan). Electrical stimulation using SEN3301 (Nihon Kohden, Shinjuku-ku, Tokyo, Japan) was applied through a pair of platinum wires, one on each side of the muscle, in physiological soft solution (150 mM NaCl, 4 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 5.6 mM glucose, 5 mM HEPES, pH 7.4, and 0.02 mM d-tubocurarine). The diaphragm was positioned midway between the two electrodes. The muscle fiber length was adjusted incrementally by using a micropositioner until peak isometric twitch force responses were obtained (i.e., optimal fiber length [L0]). L0 was measured with a microcaliper accurate to 0.05 mm. The dependence of force generation on the maximum tetanic force (P0) was assessed by stimulation frequencies of 120 pulses/s delivered in three 500-msduration trains with 2 min intervening between each train. Following these measurements, the stimulated muscle was dried and weighed after tendon attachments were removed. All forces were normalized to the dried cross-sectional area (CSA), the latter being estimated on the basis of the following formula: dried muscle weight (in mg)/[L0 (in mm) 1.06 (in mg/mm3 )]. The estimated CSA was used to determine specific tetanic forces (P0/CSA) of the muscles. Statistical analysis. Data were expressed as means SEM. After analysis of variance, if a significant F ratio was detected, comparisons among each group were performed using Fisher’s PLSD. A p value of
