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`

`Ol{JGINAL ARTICLES
`Prognostic significance of the immunohistochcmical expression of O''-methylguanine-DNJ\ methyltransferase,
`P-glycoprotein, an<l multi<lrug resistance protein-I in gliohlastomas T Nakagawa, K !do, T Sak11111a,
`fl Take11chi, K Sato and T K11hota
`Dynamics of global gene expression changes <luring brain metastasis formation N Saito, T llatori, K Aoki,
`M Hayashi, Y llirata, K Sato, II Nakayama, J llarashina, N M11rata, Z-A 7.111111g, II Nonaka, K Shi/J/lya and
`S lwab11chi
`Classic an<l <lesmoplastic me<lulloblastoma: Complete case reports and characterizations of two new cell lines
`DJ flultlw11se, PB Dallas, J Ford, V fohian, AR M11rch, M \Vatson, Ci \Vong, C !lcrtr11111, S /:'g/i, !)I, !111kcr 1111d
`UR Kees
`Transplanted human embryonic neural stem cells survive, migrate. <lilfrn:ntiatc and increasc endogcnou, nest in
`/' 7.111111g, .I l.i, }' l.iu, X Chen 1111/I (! K1111g
`expression in adult rat cortical peri-infarction zone
`Transplanted bone marrow stromal cclls improves cognitive dysfunction due to diffusc axonal injury in rats
`K Maruichi, S K11roda, Y Chiba, M llokari, I I Shichi11ohc, K llit/11 1111d )' li\'/lst1ki
`In vivo expression of proinllammatory cytokines in I IIY encephalitis: an analysis of 11 autopsy c:1,cs
`ii llayakm1·a, K /z111no, R kuhota, !:· Cic/pi, II l/111/ka 11111/ S fzw11"
`Methylation status of the MGMT gene promoter fails to predict the clinical outcome of gliohlastoma patients
`treated with J\CNU plus cisplatin C-K !'11rk, S-11 f'11rk, S-11 f.c,·, C-V Ki111, n-\\' Ki111, Sil f',l<'k, !)Ci Ki111,
`DS lleo, Ill Kim all// 11-\V J11ng
`
`II(! Xing,
`
`CASE REPORTS
`
`Diagnostic pitfall: Optic neuritis mimicking optic nerve glioma M !lerg111,111n, \VI/nick, /! Nl'llh11111·r 11111/
`S Proh.1t-Co11sin
`Coexistence of Creutzfeldt-Jakob disease, Lewy body disease. and Alzheimer\ disease: p:1thology: J\11 autopsy
`'/' lf11mgwhi, S 'frrat/11, I I lshiw, K S11k11i,
`case showing typical clinical features of Creutzfeldt-Jakob disease
`Y 1i111ahe, T Nagai,/! 711kata, K No!J11kw1i, )' !hara, T Kita1not/J and S K11rut!11
`Meningeal alveolar soft part sarcoma conlirmcd by characteristic A\'f'CIU-'/Ff:'3 fusion
`P Epaliyangc, R Gu/Ian and C Fisher
`Frontotcmporal lobar <lcgeneration with uhiquitinatcd tau-negative inclusions :ind additional 1(-synuclein
`pathology hut also unusual cercbellar uhiquitinatcd p(i2-positive,TD1'-·U-negative inclusions
`,I King,
`S AI-Sarraj and C Shaw
`Spinal cord biopsy findings of anti-aquaporin-➔ antibody-ncgative n:currcnt longitudinal 111yclitis in a p:1ticnt
`./ 1itkalw.1lti-F11jig11.111ki, S litkagi, 1' S11/w111oto a/Ill K /11utl('
`with sicca symptoms an<l hepatitis C viral infection
`Spontaneous cranial cxtra<lural hematoma: cas.; report and review of literal llrl' MF /fan 1111, fl /)/tw11ija,
`JD Pa/111cr, D lfilto11 and \V Adams
`Progressive rnulti[ocal kukoencephalopathy showing extensi1·c spinal rnrd inrnlverncnt in a patient with
`lymphocytopenia S 'fitkcda, K Vama:aki, T ,\!iyakmrn, ff 1itk11/tmlti, F lk11111 and If ,\mi
`
`//Jodi,/) ( ion:al,·:,
`
`SYl\ll'OSIUI\I: CLINICOPATIIOLOGICAL ASPECTS OF NElJIWl\llJSCULAn l>ISOIWERS -
`A NEW HORIZON
`
`,I N11ka11111r11 al/// S litlmla
`Exon-skipping therapy for Duchenne muscular dystrophy
`Usefulness of sural nerve biopsy in the genomic era
`'/' K1111da
`Pathogenesis-targeting therapeutics for spinal and bulb:n muscular atrophy (SBMA) K Suwki, M K111t11110,
`II lla111w wu/ Ci So/J/lc
`
`NEUROPATIIOLOGY EDUCATION
`
`J\n I I-year-old boy showing rapid psychomotor rcgrcssion and diffuse ccrebral white matter lesions
`YI lacltiya and M ffa1·11.1!ti
`
`Corrigendum
`
`ffiWILEY(cid:173)
`WBLACKWELL
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`This material was rn-p-ied
`at the NLM an<J may be
`5u b-j,e<t US Copyright Law s
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`379
`
`389
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`3')8
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`-+10
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`➔ 22
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`4h0
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`4KIJ
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`502
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`517
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`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Neuropathology 2009; 29, 494-501
`
`doi: I 0.1111 /j. I 440- I 789.2009.01028.x
`
`Symposium: Clinicopathological aspects of neuromuscular
`disorders - A new horizon
`Exon-skipping therapy for Duchenne
`muscular dystrophy
`
`Akinori Nakamura and Shin'ichi Takeda
`
`Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry
`(NCNP), Ogawa-higashi, Kodaira, Tokyo, Japan
`
`Duchenne muscular dystrophy (DMD) is a lethal muscle
`disorder caused by mutations in the DMD gene for which
`no mutation-targeted therapy has been available thus far.
`However, exon-skipping mediated by antisense oligonucle(cid:173)
`otidcs (AOs), which arc short single-strand DNAs, has con(cid:173)
`siderable potential for DMD therapy, and clinical trials in
`DMD patients are currently underway. This cX<m-skipping
`therapy changes an out-of-frame mutation into an in-frame
`mutation, aiming at conversion of a severe DMD pheno(cid:173)
`type into a mild phenotype by restoration of truncated
`dystrophin expression. Recently, stable and less-toxic AOs
`have been developed, and their higher efiicacy was con(cid:173)
`firmed in mice and dog models of DMD. In this review,
`we briefly summarize the genetic basis of DMD and the
`potential and perspectives of exon skipping as a promising
`therapy for this disease.
`
`Key words: antisense oligonucleotide, DMD animal model,
`DMD gene, Duchenne muscular dystrophy (DMD), dys(cid:173)
`trophin, exon skipping.
`
`INTRODUCTION
`
`Muscular dystrophy is a group of disorders that shows
`progressive muscle atrophy and weakness and the histopa(cid:173)
`thology of which reveals degeneration and regeneration of
`muscle fibers. Among them, Duchcnnc muscular dystrophy
`(DMD), an X-linked disorder, is the most common and
`produces the most severe phenotype. This disorder mani(cid:173)
`fests around the age 2-5 years by difficulty in walking, and
`the skeletal muscle involvement is progressive, resulting in
`
`Correspondence: Shin'ichi Takeda, MD, PhD, Department of Molecu(cid:173)
`lar Therapy, National Institute of Neuroscience, National Center of
`Neurology and Psychiatry (NCNP), 4-1-1 Ogawa-higashi, Kodaira,
`Tokyo 187-8502, Japan. Email: takcda@ncnp.go.jp
`Received 30 January 2009 and accepted 22 March 2009; published
`online 22 May 2009.
`
`© 2009 Japanese Society of Neuropathology
`
`patients being wheelchair-bound by the age of 13. 1l1e
`patients die of cardiac or respiratory failure due lo dilated
`cardiomyopathy around the age of 30 years, at least in
`Japan. The responsible gene, DMD, encodes dystrophin,
`which is expressed at the sarcolcmma of muscle fibers, and
`DMD mutations interrupt the reading-frame, resulting in
`a complete loss of dystrophin expression, which causes
`DMD.1 'fllc histopathology shows degeneration, necrosis,
`inflammatory cell invasion, and regeneration of muscle
`fibers, which arc eventually replaced by librous connective
`and fat tissue. Besides DMD, two phenotypes of the
`dystrophin-dclicicnt condition, Becker muscular dystrophy
`(BMD) and X-linkcd dilated cardiomyopathy (XLDCM)
`arc known. BMD is a milder variant of DMD, and XLDCM
`shows dilated cardiomyopathy without overt skeletal
`muscle signs and symptoms. /\II three phenotypes of dys(cid:173)
`trophin deficiency arc called dystrophinopathics.
`Several therapeutic strategics for treatment of DMD
`have been investigated extensively: gene therapy using
`micro-dystrophin with an adcno-associatcd virus (/\AV)
`vcctor,2 stem cell transplantation using muscle satellite
`cclls3 or bone marrow stromal cells,4 and read-through
`therapy for nonsense mutations.~ However, an effective
`treatment has not yet been established. In recent years,
`cxon skipping using antiscnsc oligonucleotidcs (/\Os) has
`been considered one of the therapeutic strategics for res(cid:173)
`toration of dystrophin expression at the sarcolcmma. AOs
`arc artificial nucleic acids that recognize a specific sequence
`of the mRNA, resulting in a change in the splicing pattern
`or translation. Currently, various A Os possessing the prop(cid:173)
`erties of high stability, high efficacy and low toxicity,
`have been developed. Herc, we review advances in exon•
`skipping therapy for DMD.
`
`THE DMD GENE AND ITS MUTATION
`
`The DMD gene is located on the human chromosome
`Xp2.1, an<l it is the largest gene in the human genome, with
`
`

`

`Exon-skipping therapy for DMD
`
`495
`
`79 exons spanning more than 2500 kb. 1l1e DMD gene
`encodes a product called dystrophin. Full-length dystro(cid:173)
`phin mRNA is about 14 kb and is mainly expressed in
`skeletal, cardiac and smooth muscles, and the brain. Dys(cid:173)
`trophin is a rod-shape structure that consists of four
`domains: (i) the N-terminal actin-binding domain; (ii) a rod
`domain composed of 24 spectrin-like rod repeats and 4
`hinges; (iii) a cysteine-rich domain that interacts with
`dystroglycan and sarcoglycan complexes; and (iv) the
`C-terminal domain that interacts with the syntrophin
`complex and dystrobrcvin. Dystrophin is localized at the
`sarcolemma and forms a dystrophin-glycoprotein complex
`(DGC) with dystroglycan, sarcoglycan, and syntrophin/
`dystrobrevin complexes. llwn, DGC links the cytoskeletal
`protein actin to the basal lamina of muscle fibers. DGC is
`considered to work as a membrane stabilizer during muscle
`contraction or a transducer of signals from the extracellu(cid:173)
`lar matrix to the muscle cytoplasm via its interactions with
`intracellular signaling molecules." Dystrophin deficiency
`leads to a condition in which the membrane is leaky under
`mechanical or hypo-osmotic stress. Consequently, Ca2
`'
`permeability is increased, and various Ca2'-depcndent
`proteases, such as calpain, arc activated in dystrophin
`deficiency. It has also been proposed that alteration of the
`expression or function of the plasma membrane proteins
`associated with dystrophin, such as neuronal nitric oxide
`synthase (nNOS), aquaporin-4, Na' channel, L-type Ca2
`'
`channel, and stretch-activated channel, arc involved in the
`molecular mechanisms of muscle degeneration.''
`In DMD patients, various mutations in the DMD gene,
`such as misscnsc, nonsense, deletion, insertion, or duplica(cid:173)
`tion, have been
`identified (http://www.hgmd.org). In
`general, when the reading-frame of amino acids is dis(cid:173)
`rupted by a mutation (out-of-frame), dystrophin is not
`expressed, resulting in the severe phenotype of DMD. On
`the other hand, when the reading-frame is maintained
`despite the existence of a mutation (in-frame), a truncated
`but still functional dystrophin is expressed, leading to the
`more benign phenotype of Becker muscular dystrophy
`(BMD). Ninety-two percent of the DMD/BMD pheno(cid:173)
`types arc explained by the "frame-shift theory." In the
`DMD gene, there arc two hot spots for mutation: around
`exons 3-7 and exons 45-55.
`
`RATIONALE OF EXON-SKIPPING
`THERAPY IN DMD
`
`In DMD, dystrophin is basically absent at the sarcolc111111a,
`although so111e dystrophin-posit ive fibers. which are called
`rcvertant fibers, arc detected in DMD patients and DMD
`animal models. The nu111ber of revertant fibers increases
`with age due lo the cycle of degeneration and rcgcnera(cid:173)
`tionY It is currently thought that the molecular 111echa-
`
`li) 2()()<) Japanese Society of Neuropathology
`
`nism underlying revertant fibers is the skipping of exon(s)
`around the original mutation, which gives rise to correction
`of the reading frame and expression of dystrophin at the
`sarcolemma.9 Consequently, exon skipping has attracted
`attention as a strategy for restoration of dystrophin expres(cid:173)
`sion in DMD.8- 10 In addition, exon-skipping therapy for
`DMD has been advanced by the development of several
`new AOs. 11 Exon-skipping therapy has been reported to be
`practical for up to 90% of DMD patients having a deletion
`13 In addition, the ethical issues involved in
`mutation. 12
`•
`exon-skipping therapy are fewer in number than those in
`gene therapy or stem-cell transplantation therapy because
`AOs are classified as a drug rather than a gene therapy
`agent by the Food and Drug Administration (FDA) of the
`USA and representative agencies in the EU and Japan.
`Based on reports that asymptomatic patients with high
`blood crcatinc kinase concentrations have an in-frame
`deletion in the DMD genc, 14.1 5 it is possible that cxon(cid:173)
`skipping therapy could convert DMD phenotype to an
`asymptomatic phenotype rather than the milder pheno(cid:173)
`type of dystrophin deficiency, BMD.
`
`DEVELOPMENT OF ANTISENSE
`OLIGONUCLEOTIDE AND DESIGN OF
`SEQUENCE
`
`Antisensc oligonuclcotides arc chemically synthesized
`20-25 base-long single-strand DNAs that are designed to
`hybridize with a complementary sequence in the target
`mRNA. In 1989, Isis Pharmaceuticals developed the AO
`drug Vitravcne (fomivirsen) for retinitis due to cytomega(cid:173)
`lovirus infection in AIDS patients, and it was the first AO
`approved by the FDA. However, the clinical application
`did not go smoothly because of adverse effects such as
`inflammation, and it was terminated in 1999.
`Various chemistries for AOs have been proposed to
`overcome the unstable nature of single-strand DNA or
`RNA molecules (Fig. I). Several modifications of AOs
`include a bicyclic-locked nucleic acid (LNA), peptide
`nucleic acid (PNA), ethylene-bridged nucleic acid (ENA),
`2'-O-methyl phosphorothionate AO (2OMeAO), phos(cid:173)
`phormliamidatc morpholino oligomer (PMO: morpho(cid:173)
`17 Development
`lino), and peptide-linked PMO (PPMO). 11
`'·
`of appropriate AOs requires consideration of several
`characteristics of AOs, such as the chemical specificity,
`affinity. nuclease resistance, stability, safety, and case of
`synthesis,1''·1~ but among them, 2OMcAO and PMO arc
`the most frequently utilized because of their suitable
`properties.
`The structure of2OMcAO is similar to that of RNA, but
`it has been methylated al the 2'-011 position of the ribose
`ring. 2OMeAO is widely used because it is relatively cheap
`to produce and easy to synthesize, has high stability and
`
`

`

`496
`
`A
`
`B
`
`C
`
`D
`
`A. Nakamura and S. fokcd11
`
`I ORbase
`b Q-base
`
`I
`-
`O=p-0
`
`0 0
`
`E
`
`F
`
`G
`
`I
`
`/
`
`o=P-N ~Orbase
`°rOrbase
`
`'-----N..---,--n-1
`O=p-N
`I
`".
`
`N
`I
`
`Fig. I Chemistries used for exon skipping. A, DNA; B, RNA; C. peptide nucleic acid (l'N/\); D, ethylene-bridged nucleic acid (ENA);
`E, 2'-0-mcthyl phosphorothionatc antiscnsc oligonuclcoti<lc (20Mc/\0); r, phosphorodiamicJatc morpholino oligomer (l'MO) (mor(cid:173)
`pholino), G, peptide-linked PMO (l'l'MO).
`
`affinity to mRNA, and is also resistant to nucleases.
`However, the low solubility of20MeAO prevents its use at
`higher dosages. 19
`PMO has a morpholine ring instead of a deoxyribose
`ring in DNA or ribose ring in RNA, and the morpholinc
`rings bind to each other through phosphorodiamidatc
`instead of phosphoric acid. PMO is non-ionic, which
`minimizes protein interactions and nonspecific antisense
`effects, and it has several advantages such as high solubility
`in water and high binding capacity to mRNA. PMO docs
`not stimulate or activate either Toll-like receptors or
`inflammatory responses mediated by interferon or Nf.-KI3.
`AVI I3iopharma Inc. (Corvallis, OR, US), which is the only
`pharmaceutical company that currently produces a Good
`Manufacturing Practice grade of PMO, has promoted
`several clinical trials targeting cardiovascular restcnosis
`(Phase 11, finished), hepatitis C virus (Phase I, finished),
`Ebola virus (Phase I, ongoing), Marburg virus (Phase I,
`ongoing), and rheumatoid arthritis (Phase I, ongoing). To
`improve the stability of !'MO in blood and cells and to
`
`increase the uptake of !'MO into the cells, cell-penetrating
`peptides (Cl'l's) such as II-alanine (B), II-arginine (R). or
`6-aminohcxanc (X) arc added, and the compound is called
`a peptide-conjugated l'MO (l'l'MO).
`Designing the sequence of an J\O to skip a particular
`cxon in the splicing process from premature mRNJ\ to
`2° Knowledge of the molecular
`mRNJ\ is very important. 11
`'·
`mechanism of splicing tells us that some proteins and
`splicesome complexes are involved in the splicing machin(cid:173)
`ery through an exon-intron consensus sequence or an
`cxon splicing enhancer (ESE). /\Os target the exon-intron
`boundaries or the ESE and subsequently can inhibit
`the binding of the spliceosome to premature mRNA.
`However, when web-based software, such as ESElinder
`(http://rulai.cshl.edu/tools/ESE), is used to design an AO
`sequence to target an ESE, exon-skipping is not always
`22 An AO targeting a non-ESE scquencc such
`induced.21 •
`as exon-intron boundary sequences can often cffcctivcly
`induce exon skipping, but the effccts of thc samc J\O differ
`in vilro and may also differ i11 1fro. Reccntly. \\'cc ct al.
`
`© 2()()() Japancsc Socicty of Ncuropathology
`
`

`

`Eron-skipping therapy for /J/1'! /J
`
`497
`
`developed bioinformatics tools to optimize AO sequences
`based on the prc-mRNA secondary structure. 23
`
`vectors and finding a way to prevent immune responses
`t.lue to subsequent injections of AAV vectors needs to be
`addressed.
`
`EXON SKIPPING IN DMD ANIMAL
`MODELS
`
`Before application of AOs to exon skipping in DMD, in
`vitro and in vivo studies using aninrnl models are indispens(cid:173)
`able. Cultured skeletal muscle cells derived from DMD
`patients are often used to evaluate exon-skipping effi(cid:173)
`ciency.2rn However, in vitro studies are limited because
`while we can examine the effectiveness of skipping itself,
`we cannot evaluate the functional repair in an in vitro
`system. On the other hand, animal models can be used to
`assess the efficacy of cxon skipping as well as the improve(cid:173)
`ment of muscle function. In this section, studies of the
`DMD mouse models mt!x and 1111lx52, which we estab(cid:173)
`lished, and dystrophic dog will be described.
`
`M<lr mouse
`·111c 111!1.r mouse has a nonsense mutation in cxon 23 of the
`OM/J gene, resulting in loss of dystrophin. 'TT!is mouse
`shows a mild but non-progressive muscle weakness of the
`limbs, although progressive muscle degeneration, necrosis
`and fibrosis occur in respiratory muscles including the dia(cid:173)
`phragm.21' Lu et al. reported the local administration of
`2OMcAO with the non-ionic polymer rt27, which pro(cid:173)
`motes intracellular uptake of 2OMcAO, to the skeletal
`muscles of 2-wcck-old nulx mice. ·n1c result showed that
`dystrophin together with 13-dystroglycan, sarcoglycans, and
`nNOS was restored in 20% of the muscle fihcrs.22 further(cid:173)
`more, systemic administration of the anti-sequences of the
`same 2OMcAO with Fl27 revealed that dystrophin was
`expressed in the skeletal muscle of the whole body except
`the heart. 'Illcre was no toxicity of 2OMcJ\O, but the
`expression did not reach a therapeutic lcvcl. 21 Wells ('t al.
`reported that local administration of 2OMeJ\O using clcc(cid:173)
`troporation restored dystrophin expression to up to 20%
`of the normal levcl. 27 Systemic induction of dystrophin
`expression by l'MO administration has reached a feasible
`level in whole body skeletal muscle, although not the
`hearl.'11·2' In addition, the more recently developed l'l'MO
`induced high expression of dystrophin in the heart as well
`in whole body skeletal muscles.''' A unique cxon-skipping
`method was proposed in which the mutated exon 23 on the
`rnllNJ\ of 11ulx mice is removed by a single administration
`of an J\J\ V vector expressing antiscnsc scquc11<.:cs linked to
`a modified U7 small nuclear RNA. "1 This may teach us a
`method to prevent repeated injections of AO. C.oycnvallc
`<'I al. indeed showed a strong and long-term recovery of
`t.lystrophin expression and improverm:nt of muscle func(cid:173)
`tion in 11/(IX mice, "1 hut the issue oft he cytotoxicity of A AV
`
`ri) 200<J .Japanese Society of NL'llf'llpathology
`
`Dystrophic dog
`Muscular dystrophy in dogs was originally identified in
`retriever
`golden retrievers and designated "Golden
`muscular dystrophy" (GRMD). GRMD shows progressive
`skeletal muscle weakness and atrophy as well as abnormal
`electrocardiographic findings and myocardial fibrosis, like
`those seen in DMD. However, the dogs are too large to be
`maintained conveniently, so we have established a colony
`of medium-sized beagle-based dystrophic dogs (canine
`X-linkcd muscular dystrophy in Japan: CXMD1) at the
`National Center of Neurology and Psychiatry, Tokyo, by
`using artificial insemination of frozen GRMD semen. 31 The
`level of scrum crcatine kinase in CXMD1 is very high soon
`arter birth, ant.I about 25-33% of the pups die of respira(cid:173)
`tory failure during the neonatal pcriot.l. Around the age of
`2-3 months, atrophy and weakness of limb muscles appear,
`then the dogs develop gait disturbance, joint contracture,
`macroglossia, and dysphasia. Those symptoms rapidly
`progress until the dogs arc 10 months of age, and then the
`progression is retardcd. 32 CXMD1, GRMD, and DMD
`have similar cardiac involvement, including distinct deep
`Q-waves on the electrocardiogram and fibrosis of the left
`ventricular wall. 3.1 The distinct deep Q-waves arc ascribed
`to fibrosis in the postcrobasal region of the left ventricular
`wall in DMD, but we found that the deep Q-wavcs on
`cchocardiograms precede the development of histopatho(cid:173)
`logically apparent fibrosis in CXMD1. 33 When we investi(cid:173)
`gated the cardiac pathology of CXMD1, we found that the
`Purkinjc fibers showed remarkable vacuolar degeneration
`t.lcspite the absence of detectable fibrotic lesions in the
`ventricular myocardium. TI1c degenerated Purkinjc fibers
`were coincident with ovcrcxprcssion of Dp71, a C-tcrminal
`truncated isoform of dystrophin, at the sarcolcmma and
`translocation of calcium-dependent protease p-calpain to
`the cell periphery near the sarcolcmma or in the vacuoles.
`Utrophin, a homologue of dystrophin, was highly upregu(cid:173)
`latcd in the l'urkinjc fibers in the early stage, but the
`expression was dislocated when vacuolar degeneration was
`recognized at 4 months of age.-'' The selective degeneration
`of J>urkinjc lihcrs can he associated with distinct deep
`O-waves on electrocardiograms and the fatal arrhythmia
`seen in dystrophinopathy. Thus. the dystrophic dog is a
`useful model to examine pathogenesis and therapeutic
`strategics because the phenotype and genetic background
`arc closer to human DMD than those of the mouse model.
`'Ilic dystrophic dogs have a point mutation at the intron
`<i splice acceptor site in the canine /JAIi) gene, resulting in
`skipping of cxon 7. A prcmatmc stop codon arises in exon
`
`

`

`498
`
`A. Nak1111111ra and S. 'fokedc,
`
`8 and dystrophin is not produced. Recently, we and
`researchers at the Children's National Medical Center in
`the USA used three PMOs targeting cxons 6 and 8 to
`convert an out-of-frame mutation into an in-frame muta(cid:173)
`tion, and mixtures of the PMO were systemically adminis(cid:173)
`35 The result showed that dystrophin was
`tered to CXMD1•
`restored in the entire body skeletal muscle except the heart
`but that heart muscle function was improved. llms, we
`showed for the first time that multi exon-skipping is fea(cid:173)
`sible in vivo. Another study reported that PPMO had the
`highest efficacy in restoration of dystrophin expression
`when the effectiveness of exon skipping of 2OMeAO,
`PMO, and PPMO was compared in GRMD muscle cclls.36
`
`Mdx52 mouse
`
`Katsuki and his colleagues generated another DMD
`mouse model, mdx52, in which exon 52 of the murinc
`DMD gene was deleted hy using a homologous recombi(cid:173)
`nation techniquc.37 Like the mdx mouse, mdx52 lacks dys(cid:173)
`trophin and presents dystrophic changes including muscle
`hypertrophy. In particular, the retina-specific dystrophin
`isoform Dp260 is absent and abnormal clcctrorctino(cid:173)
`graphic findings were dctcctcd. 38 Recently, we tried cxon
`51-skipping using PMO in nulx52 mice to convert an out(cid:173)
`of-frame mutation into an in-frame mutation and found
`restoration of dystrophin expression in various muscles
`and improvement of muscle pathology and function (Aoki
`et al., unpublished data).
`
`PROSPECTS FOR CLINICAL TRIALS
`FOR DMD
`
`It is thought that the number of patients having the same
`nonsense or deletion mutation of cxon 23 as the nulx
`mouse or the same deletion of exon 7 as the dystrophic dog
`is very small. To provide exon-skipping therapy to more
`patients, it will he necessary to target the hot spots of
`mutation in the DMD gene.Among the mutation hot spots,
`patients having one and more deletions within cxons 45-55
`account for 60°/4, of DMD patients having deletion muta(cid:173)
`tions, making this area a prominent target. 'Il1is section
`reviews the preclinical studies of cxon skipping targeting
`one cxon (single-cxon skipping) and multiple cxons (multi(cid:173)
`exon skipping) within cxons 45-55. Herc, the nulx52 mouse
`is an indispensable animal model for the investigation of an
`exon-skipping strategy targeting this hot spot region.
`
`Single-exon-skipping therapy
`
`Based on the Leiden Muscular Dystrophy database (http://
`www.dmd.nl), exon skipping targeting cxon 51 may be
`applicable to about 15'¼, of patients with DMD having a
`deletion mutation. In the Netherlands, clinical trials of
`
`cxon 51-skipping therapy for patients with the deletion or
`either cxons 48-50, cxons 49-50, cxon 50, or cxon 52 have
`been conducted. 3
`1 In that study, 2OMcAOs were injected
`'
`into the tihialis anterior (TA) muscle of the patients, and
`the efficacy of cxon skipping, restoration of dystrophin
`expression, and improvement of MRI findings were
`reported, although the TA muscle function could not be
`evaluated because of local administration. Based on our
`on-going study of exon 51 skipping in the mdx52 mouse,
`a certain degree of improvement of muscle function is
`expected (Aoki et al., unpublished data). In addition, the
`UK 40 and US/Japan arc planning clinical trials of exon
`51-skipping therapy using PMO.
`In the Leiden Muscular Dystrophy database, relatively
`small-sized in-frame deletions, mainly including cxons 50
`and 51 ( e.g. deletion of cxons 45-51, 47-5 I, 48-51, 49-51,
`50-51, 51, or 52) cause a high rate of DMD phenotypes
`(56-100%) rather than the BMD phcnotypc.41 'll1e mecha(cid:173)
`nism underlying the high ratio of DMD phenotypes despite
`the in-frame mutation remains unclear, but the hinge 3
`region coded by cxons 50-51 of the !)Ml) gene might have
`an important functional role in dystrophin. On the other
`hand, larger deletions including cxons 50-51 ( e.g. deletion
`of cxons 45-53 or 45-55) more frequently present with a
`BMD phcnotypc.41 Tlrns, hinge 3 might bestow flexibility
`on the protein structure, and when hinge 3 is absent, the
`longer spans between hinges might become fragile to
`mechanical stress. However, we also need to point out that
`gene deletion should be confirmed not only on a genomic
`DNA level but also an mRNA level.
`
`Mnlti-exon-skipping therapy
`
`As described above, exon 51-skipping therapy may be fea(cid:173)
`sible for up to 15% of DMD patients having a deletion
`mutation, but this therapeutic method remains a "custom(cid:173)
`madc therapy," and large numbers of DMD patients will
`not benefit from it. We recently reported three unrelated
`patients with a deletion of cxons 45-55, which covers the
`entire hot spot region, who have very mild skeletal muscle
`involvement and can still walk unassisted late in lifc. 4c
`Ucroud et al. also described 15 patients having an cxon
`45-55 deletion who had very mild or asymptomatic skeletal
`muscle involvcmcnt.41 Furthermore, when we examined
`the number of patients having deletions within cxons 45-55
`in the Leiden Muscular Dystrophy database combined
`with previous data,42.4 1 the