Review Article. Duchenne and Becker Muscular Dystrophy: From Gene Diagnosis to Molecular Therapy

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1 IUBMB Life, 53: , 2002 Copyright c 2002 IUBMB /02 $ DOI: / Review Article Duchenne and Becker Muscular Dystrophy: From Gene Diagnosis to Molecular Therapy Masafumi Matsuo Division of Molecular Medicine, Kobe University Graduate School of Medicine, Chuo, Kobe, , Japan Summary Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed. IUBMB Life, 53: , 2002 Keywords DMD/BMD; dystrophin; mutation; splicing; treatment. INTRODUCTION Duchenne muscular dystrophy (DMD) is a common inherited disease with a worldwide incidence of 1 in 3,500 male births. DMD is a lethal disorder of childhood usually associated with a functional deficiency of dystrophin. The affected individuals are wheelchair-bound by the age of 12 and succumb to cardiac or respiratory failure in the mid to late 20s. Interestingly, a milder form of the disease called Becker muscular dystrophy (BMD) is distinguished from DMD by delayed onset, later dependence on wheelchair support, and longer life span. Received 4 December 2001; accepted 6 March Address correspondence to Masafumi Matsuo, Division of Molecular Medicine, Kobe University Graduate School of Medicine, Kusunokicho, Chuo, Kobe, , Japan. Fax: matsuo@kobe-u.ac.jp After the submission of this manuscript, it was reported that negamycin, a dipeptide antibiotic, was able to induce a read-through effect to express dystrophin in the mdx mouse (29). Because negamycin has low toxic side effects compared with gentamycin, this therefore may be more suitable for clinical use. The gene defective in DMD/BMD was isolated in 1986 and was named dystrophin. Since then, diagnosis of DMD/BMD has been done at the molecular level, leading to a better understanding of the disease process. Consequently, studies regarding the application of molecular therapy have been promoted. Present progress made in this area of research, with particular reference to dystrophin Kobe and molecular therapy for DMD, is presented. Gene Diagnosis Both DMD and BMD are allelic disorders caused by mutations of the dystrophin gene. The dystrophin gene is 3,000 kb in size and consists of 79 exons encoding a 14-kb mrna (1). The unusually high incidence of DMD/BMD in all human populations could be simply a reflection of the enormous mutation target size of the gene but the recombination rate is reported to be four times the rate expected for a gene of this size (2). To identify such mutations, segments of cdna have been used as probes to specifically examine all the exons by Southern blot analysis. Most of the identified mutations are deletions with over 65% of patients exhibiting the loss of one or more exons at the genomic DNA level. Two deletion hot spots have been identified near the 5 end and in the central region of the dystrophin gene, respectively. Duplications have also been identified in a small number of patients. Currently PCR amplification of 19 deletion prone exons (exons 1, 3, 4, 6, 8, 12, 13, 17, 19, 43, 44, 45, 47, 48, 49, 50, 51, 52, and 60) is commonly used to screen deletion mutations and nearly half of all cases are shown to have deletion mutations. In the remaining cases, identification of the causative mutation remains a laborious target due to the difficulty in detecting a single point mutation in the 3,000-kb gene. Dystrophin mrna, which is 100 times smaller than the dystrophin gene consisting of more than 99% introns, has been analyzed to facilitate the identification of mutations in the dystrophin gene (3). Analysis of dystrophin mrna expressed in lymphocytes leads not only to the identification of rare genomic 147

2 148 MATSUO mutations but also to disclosures of nonauthentic splicing (4, 5). As a result, more than 100 point mutations have been reported ( In the advent of recent advances in mutation analysis techniques, more than 90% of DMD cases are shown to have mutations in the dystrophin gene (6, 7). Reading-Frame Rule Although both DMD and BMD patients have been shown to have deletion mutations, the extent of the deletion does not always correlate with the severity of the disease: some BMD patients with mild symptoms have deletions encompassing numerous exons whereas some DMD patients with severe symptoms lack only a few exons. In some cases, long deletions resulting in BMD and short deletions resulting in DMD may even overlap. According to the reading-frame rule (8), BMD patients with long deletions may be able to produce a dystrophin mrna that would still direct the production of an internally truncated semifunctional protein. Shorter deletions harbored by severe DMD patients, on the other hand, would bring together exons that, when spliced, could change the translational reading frame in the mrna such that a premature stop codon is created. This rule predicts that milder BMD patients would produce a smaller semifunctional protein, whereas DMD patients would either produce a severely truncated form lacking the entire C-terminal region or would not produce a protein at all. Subsequent gene analyses have shown that over 90% of deletion mutations that cause BMD maintain the dystrophin mrna reading frame, whereas those causing DMD are usually frame shifts. Accordingly, point mutations identified in DMD are nonsense mutations (7) except rare DMD cases with missense mutations (9, 10). Immunohistochemical analyses have demonstrated that dystrophin is present in muscle cell membranes. As expected, this protein is completely missing in boys with DMD, whereas muscle tissue from BMD patients contains reduced amounts of dystrophin. Thus, DMD and BMD represent examples of allelic heterogeneity. Western blot analyses using dystrophin antibodies have revealed a band corresponding to 427 kda, close to the predicted size of dystrophin, in extracts of normal muscle tissues. Shorter proteins can sometimes be detected in extracts of tissues from patients with BMD, yet no protein can be detected in DMD. Dystrophin Kobe In one particular dystrophin gene mutation named dystrophin Kobe (11), we found that exon skipping during splicing was induced by the presence of an intra-exon deletion mutation in the genome although all of the consensus sequences known to be required for splicing were unaffected (12). The deletion was detected by PCR analysis which revealed that the product amplified from the DMD case in question was smaller than normal. This result suggested the presence of a novel mutation within the amplified region. Sequence analysis confirmed that this was the case by showing that 52 bp out of 88 bp of exon 19 were deleted at 2 3 bp upstream from the splice donor site. This 52-bp deletion was considered to result in a frame-shift mutation that would cause DMD. The dystrophin mrna of dystrophin Kobe was then analyzed using reverse-transcription PCR (RT-PCR). Surprisingly, the product amplified from the region encompassing exon 19 was smaller than the predicted one according to the results of the genomic DNA analysis. Sequence analysis indicated that the whole of exon 19 was missing from the dystrophin cdna causing an out-of-frame mutation. This indicated that the deletion mutation within an exon sequence could induce a splicing error during the maturation of messenger RNA even though the known consensus sequences at the 5 and 3 splicing sites of exon 19 were maintained (12). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. An in vitro splicing system using artificial dystrophin pre-mrnas disclosed that splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19 (13). It was next investigated whether antisense oligonucleotides against the deleted sequence modulates splicing. An antisense 31-mer 2 -O-methylribonucleotide complementary to the 5 half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mrna in a dose- and time-dependent manner (13). These results indicated that the deleted region is a splicing enhancer sequence that is necessary for proper splicing of intron 18 even in the presence of splicing consensus sequences. Since the aforementioned result suggested a possibility of artificial induction of exon 19 skipping, the antisense oligonucleotide against the splicing enhancer sequence was then transfected to normal lymphoblastoid cells. Remarkably, exon 19 sequence in all dystrophin mrnas disappeared 24 h after the transfection (14). The antisense oligonucleotide was thus proved to be a powerful tool with the ability to induce exon 19 skipping. Production of Dystrophin in DMD-Derived Muscle Cells As the injection of dystrophin gene into muscle will not be feasible for some time, an alternative strategy for DMD treatment might be to retard the progression of the clinical symptoms, i.e., to convert the DMD into the BMD phenotype. Theoretically, this therapy can be done by changing a frame-shift mutation, causing DMD into an in-frame mutation characteristic of BMD by modifying the dystrophin mrna. Because transfection of the oligonucleotide against a splicing enhancer sequence of exon 19 has been shown to induce exon 19 skipping, we subsequently investigated whether the antisense nucleotide can be used to treat a DMD case with a 242-nucleotide deletion of exon 20 (Fig. 1). If exon 19 (88 bp) skipping can be induced in this case, the translational reading frame of dystrophin mrna will be restored. As a result, this modulation of splicing should lead to the production of internally deleted dystrophin in muscle cells from the case. A Japanese DMD patient was identified to have a deletion of exon 20 of the dystrophin gene. Primary muscle culture cells established from his muscle were

3 DUCHENNE AND BECKER MUSCULAR DYSTROPHY 149 This prompted us to examine the possibility whether the intravenous injection of antisense oligonucleotide was a good means of delivery into skeletal muscle. Fluorescence-labeled oligonucleotide was thus injected into the peritoneum of mdx mice and nuclei of muscle cells were noted to be fluorescent positive. Remarkably exon 19 skipping was observed in cardiac and skeletal muscle (17). It thus can be concluded that the antisense oligonucleotide can be delivered to muscle cells by directly injecting into the venous system. Figure 1. Correction of the translational reading-frame using antisense oligonucleotide. In a DMD case with an exon 20 deletion (242 bp), splicing of the dystrophin pre-mrna proceeds to produce an out-of-frame mrna lacking exon 20 (lower diagonal lines). In the presence of antisense oligonucleotide against the splicing enhancer sequence (bold bar), the recognition of exon 19 (88 bp) was inhibited and splicing proceeds from the 5 end of intron 18 to the 3 end of intron 20, thereby producing an in-frame mrna (upper bold diagonal lines). Boxes and number in boxes indicate exon and exon numbers, respectively. The shaded box is exon 20 that is deleted in genomic DNA. Arrows indicate splicing directions. transfected with the 31-mer-phosphorothioate oligonucleotide (5 -GCCTGAGCTGATCTGCTGGCATCTTGCAGTT-3 )covering a splicing enhancer sequence of exon 19. Introduction of the oligonucleotide into the nuclei of cultured cells led to skipping of exon 19 in a proportion of total mrna. The simultaneous disappearance of consecutive exons 19 and 20 from the dystrophin mrna restored the translational reading frame, removed a downstream premature stop codon from the mrna, and caused the production of an in-frame mrna. As expected, dystrophin-positive cells were identified. The percentage of dystrophin positive cells was nearly 20% at the 10th day after transfection (15). Our result is the first to show that oligonucleotided against a splicing enhancer sequence can successfully induce exon skipping in DMD muscle cells and can lead to the production of an internally deleted dystrophin. This result paves a novel way to treat DMD patients by administering an oligonucleotide against a splicing enhancer. One last question that needed to be answered before initiating the actual treatment of DMD with the antisense oligonucleotide was how the oligonucleotide was to be delivered to muscle cells. Transfection of the virus vector into every skeletal muscle cell has frequently resulted in failure and this is the main subject of study in gene therapy to establish a way to transfect into muscle cells. Recently it was shown that Evans blue, a low molecular weight dye, was directly diffused into damaged muscle cells of mdx mice (16). Molecular Therapy for DMD The main aim of DMD/BMD gene therapy is to establish a way to inject constructed dystrophin genes consisting of partial or full-length cdna joined to an appropriate promoter. Although much progress has been made in this field of study, we still seem to be a long way from achieving a clinically significant result. As an alternative for gene-transfection, molecular therapies including antisense oligonucleotide treatment for DMD have been studied (15, 18, 19). Here, not only antisense oligonucleotide treatment but also short fragment homologous replacement, mismatch repair, and translational readthrough treatments are reviewed. Antisense Oligonucleotide. As mentioned previously, exon skipping induced by antisense oligonucleotides has been studied. Three different targets were examined to block its proper function by antisense oligonucleotides; splicing enhancer sequence, exonic purine-rich sequence, and splicing consensus sequence (15, 18, 19). Antisense oligoribonucleotides have been used to modify the processing of dystrophin pre-mrna in the mdx mouse, a model of DMD caused by a nonsense mutation in exon 23. By targeting 2 -O-methyl antisense oligoribonucleotides to block splicing consensus sequences involved in normal dystrophin pre-mrna splicing, excision of exon 23 and the nonsense mutation without disrupting the reading frame was induced. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes (18). Ten days after the injection, 10% of skeletal muscle cells became dystrophin positive. Blocking of the splicing consensus sequence with antisense oligonucleotides is a popular candidate approach. However, the splicing consensus sequence is common to all exons in human genes and there is concern that the sequence may trigger dysfunctional splicing of a non-target exon. Because several exons have been shown to include purinerich regions, which are necessary for splicing of the upstream intron, these sequences are also likely targets for antisense oligonucleotides for the induction of exon skipping. Recently, the antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin has been reported (19), considering that exon 45 is the single most frequently deleted exon in DMD and that exon ( ) deletions cause only a mild form of BMD. Among

4 150 MATSUO Table 1 Molecular therapy for DMD Target Effect Dystrophin Author 1. Antisense oligonucleotide mdx mouse Splicing consensus sequence Exon 23 skipping Positive Mann et al. (18) Human cells Splicing enhancer sequence Exon 19 skipping Positive Takeshina et al. (17) Human cells Poly-purine sequence Exon 46 skipping Positive van Deutekom et al. (19) Method Dystrophin Author 2. Short-fragment homologous replacement mdx mouse Double stranded DNA Positive Kaspa et al. (22) 3. Mismatch repair mdx mouse Chineric RNA/DNA oligonucleotide Positive Rando et al. (23) Retriever Chineric RNA/DNA oligonucleotide Positive Bartlett et al. (24) Drug Dystrophin Author 4. Translational readthrough mdx mouse Gentamicin Positive Barton-Davis et al. (26) Human in vivo Gentamicin Negative Howard et al. (25) mdx mouse Negamycin Positive Arakawa et al. (29) several oligonucleotides against the purine-rich sequence of exon 46, one sequence showed the strongest activity to block splicing. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the antisense oligonucleotides induced skipping of exon 46 in only approximately 15% of the mrna and led to normal amounts of properly localized dystrophin in at least 75% of myotubes (19). These indicated that the purine-rich sequence could be blocked to induce exon skipping. Disruption of the splicing enhancer sequence to induce exon skipping, as mentioned previously, was further evidenced by the fact that a nonsense mutation the exon 27 the dystrophin gene resulted in exon 27 skipping, producing an in-frame dystrophin mrna (20). The basic mechanism of exon skipping was shown, by in vitro experiments, to be due to the loss of the splicing enhancer function arising from a single nucleotide error in the exon 27 sequence. In addition, another natural example causing the conversion of DMD to BMD was identified in a nonsense mutation of exon 29 (21). Although the basic mechanism of exon skipping was not examined in this case, it is believed that the mutation would disrupt the splicing enhancer sequence. Short-Fragment Homologous Replacement. Targeted genetic correction of mutations is a potential strategy for treating nonsense mutations. One method of targeted gene repair is shortfragment homologous replacement. The application of a doublestranded method of short-fragment homologous replacement to the mdx mouse was investigated in vitro and in vivo by Kapsa et al. (22). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition in cultured myoblasts and in the tibialis anterior muscles from male mdx mice. Multiple transfection and variation of lipofection reagent both improved the in vitro short-fragment homologous replacement efficiency. Successful conversion of mdx to the wild-type nucleotide was achieved in 15 to 20% of cultured loci and in to 0.1% of tibialis anterior muscles. The genetic correction of mdx myoblasts was shown to persist up to 28 days in culture and for at least 3 weeks in tibialis anterior muscles. It was thereby proposed that short-fragment homologous replacement might find some application in DMD with further improvements to in vitro and in vivo gene repair efficiencies. Mismatch Repair. Chimeric RNA/DNA oligonucleotides ( chimeraplasts ) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The feasibility of chimeraplast-mediated gene therapy was tested for muscular dystrophies (23). A chimeraplast designed to correct the point mutation in the dystrophin gene in mdx mice was directly injected into muscles. Immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers in any muscle represented 1 2% of the total number of fibers in that muscle. Ten weeks after the single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 weeks, indicating that the expression was stable. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach for muscular dystrophies. The efficiency in correcting mutations using chimeric RNA/ DNA oligonucleotides has been examined in the canine model of DMD in golden retrievers (GRMD) in which a point mutation within the splice acceptor site of intron 6 leads to the deletion of exon 7 from the dystrophin mrna and the consequent

5 DUCHENNE AND BECKER MUSCULAR DYSTROPHY 151 frameshift causes the early termination of translation. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a 6-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated the in vivo repair of the mutation, the synthesis of the normal-sized dystrophin product and positive localization to the sarcolemma. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscles of a live animal using a chimeric oligonucleotide (24). Translational Readthrough of Stop Codon. Aminoglycoside antibiotics are currently being tested for the efficacy in the treatment of DMD carrying a nonsense mutation in the dystrophin gene as a result of their ability to induce a translational readthrough of stop codons (25). In the mdx mouse, gentamicin has been shown to suppress truncation of the protein and to ameliorate the phenotype (26). 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