NEUROLOGY
`
`WWW.ARCHNEUROL.COM
`JANUARY 2009
`
`A renaissance for antisense oligonudeotide drugs in neurology: Exon skipping breaks new ground. Mechanism of multiexon skipping
`of ex45-55 to rescue 60% of DMD patients wit11 dystrophin deletions. See page 32.
`
`Defining Multiple Sclerosis Treatment Response
`With Magnetic Resonance Imaging
`P. S. Giacomini, M D, FRCP(C), et 11l
`
`Imaging Correlates of Leukoc yte Accumulation and
`CXCR4/CXCL12 in Multiple Sclerosis
`N. M . Moll, MD, PhD, et al
`
`Translational Research in Epilepsy Genetics
`S. A. M ullen, MBBS, and I. E. Scheffer, MBBS, PhD
`
`A Renaissance fo r Antisense Oligonucleotide
`Drugs in Neurology
`T. Yokota, PhD, et al
`
`Increased Relapse Rate in Pediatric-Onset
`Compared With Adult-Onset Multiple Sclerosis
`M . P. Gorm a11, MD, et tl[
`
`Carbon 11- Labeled Pittsburgh Compound Band
`Carbon 11- Labeled (R)-PK11195 Positron Emission
`Tomographic Imaging in Alzheimer Disease
`C. A. Wiley, MD, PhD, et al
`
`ARCHIVES EXPRESS Heterogeneity in Response
`-
`-
`to Interferon Beta in Patients
`With Multiple Sclerosis
`A . W Chiu, BS, et al
`
`Personality Development in the Context of
`Intractable Epilepsy
`S. f. Wilson, PhD, et al
`
`COM PLETE T AlJLE OF C ONTENTS ON PAGE 7
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`JAMA
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`ARCHIVES
`NEUROLOGY
`
`JANUARY 2009
`
`VOLUME 66, NUMBER l
`
`PAGES 1-144
`
`This issue will be available onlinejanuary U , 2009.
`
`EDITORIAL
`
`Defining Multiple Sclerosis Treatment Response
`With Magnetic Resonance Imaging:
`How Much Activity ls Too Much?
`Paul S. Giacomini, MD, FRCP(C); Douglas L Arnold, MD;
`Amit Bar-Or, MD, FRCP(C), MSc;JackP. Antel, MD
`
`NEUROLOGICAL REVIEWS
`
`Translational Research in Epilepsy Genetics: Sodium
`Channels in Man to lntemeuronopatby in Mouse
`Saul A. Mullen, MBBS; Ingrid E. Scheffer, MBBS, PhD
`
`A Renaissance for Antisense Oligonucleotide Drugs
`in Neurology: Exon Skipping Breaks New Ground
`Toshifumi Yokota, PhD; Shin'ichi Takeda, MD, PhD;
`Qi-Long Lu, MD, PhD; Terence A. Partri.dge, PhD;
`Akinori Nakamura, MD, PhD; Eric P. Hoff man, PhD
`
`ORIGINAL CONTRIBUTIONS
`
`Heterogeneity in Response to Interferon Beta
`in Patients With Multiple Sclerosis:
`A 3-Ycar Monthly Imaging Study
`Annie W. Chiu, BS; Nancy Richert, MD, PhD;
`Mary Ehrmantraut, MS;_Joan Ohayon, MSN;
`Shiva Gupta, MD; Giuseppe Bomboi, MD;
`Deeya Gaindh, AB; Fredric K. Cantor, MD;
`Joseph A. Frank, MS, MD; Henry F. McFarland, MD;
`Francesca Bagnato, MD, PhD
`
`Imaging Correlates of Leukocyte Accumulation
`and CXCR4/CXCL12 in Multiple Sclerosis
`Natalia M. Moll, MD, PhD; Michael B. Cossoy, MD;
`Elizabeth Fisher, PhD; Susan M. Staugaitis, MD, PhD;
`Barbara H. Tucky, BS; Anna M. Rietsch, BS;
`Ansi Chang, MD; Robert]. Fox, MD;
`Bruce D. Trapp, PhD; Richard M. Ransohoff, MD
`
`Increased Relapse Rate in Pediatric-Onset
`Compared With Adult-Onset Multiple Sclerosis
`Mark P. Gorman, MD; Brian C. Healy, PhD;
`Mariann Polgar-Turcsanyi, MS; Tanuja Chitnis, MD
`
`Carbon 11-Labeled Pittsburgh Compound B
`and Carbon 11-Labeled (R)-PK11195
`Positron Emission Tomographic Imaging
`in Alzheimer Disease
`Clayton A. Wiley, MD, PhD;
`B1ia11]. Lopresti, BS; Snram Venneti, MD, PhD;
`Julie Price, PhD; William E. Klunk, MD, PhD;
`Steven T. DeKosky, MD; Chester A. Mathis, PhD
`
`19
`
`21
`
`32
`
`39
`lCMEl
`
`44
`
`54
`
`60
`
`Personality Development in the Context
`of Intractable Epilepsy
`Sarah]. Wilson, PhD; Joanne M. Wrench, PhD;
`Anne M. McIntosh, PhD; Peter F. Bladin, MD;
`Samuel F. Berkovic, MD
`
`Neoplastic Meningitis-Related Prognostic
`Significance of the Karnofsky Performance Status
`Marc C. Chamberlain, MD;
`Sandra K. Johnston, PhD, RN; Mike]. Glantz, MD
`
`68
`
`74
`
`Association of Subdural Hematoma With Increased
`Mortality in Lobar Intracerebral Hemorrhage
`Pratik V. Patel, MD; Emilie FitzMaurice, AB;
`R. N. Kaveer Nandigam, MD; Pavan Auluch, MD, PhD;
`Anand Viswanathan, MD, PhD;Joshua N. Goldstein, MD, PhD;
`Jonathan Rosand, MD, MS; Steven M. Greenberg, MD, PhD;
`Eric E. Smith, MD, MPH, FRCPC
`
`79
`
`85
`
`92
`
`97
`
`Protean Phenotypic Features of the A3243G
`Mitochondrial DNA Mutation
`Petra Kaufmann, MD, MSc; Kristin Engelstad, BS;
`Ying Wei, PhD; Romana Kulikova, MD;
`Maryam Oskoui, MD; Vanessa Battista, MS, CPNP;
`Dorrns Y. Koenigsberger, MSN, CPNP;
`Juan M. Pascual, MD, PhD; Mary Sano, PhD;
`Michio Hirano, MD; Salvatore DiMauro, MD;
`Dikoma C. Shungu, PhD; Xiangling Mao, MS;
`Darryl C. De Vivo, MD
`
`Olfactory Function in Corticobasal Syndrome
`and Frontotemporal Dementia
`Matteo Pardini, MD; Edward D. Huey, MD;
`Alyson L. Cavanagh, BS;Jordan Grafman, PhD
`
`Episodic Ataxia Associated With EAATl
`Mutation C186S Affecting Glutamate Reuptake
`Boukje de Vries, MSc; Hafsa Mamsa, MSc;
`Anine H. Stam, MD;]ijun Wan, PhD;
`Stef L M. Bakker, MD, PhD; Kaate R.]. Vanmolkot, PhD;
`Joost Haan, MD, PhD; Gisela M. Terwindt, MD, PhD;
`Elles M.]. Boon, PhD; Bruce D. Howard, MD;
`Rune R. Frants, PhD; Robert W. Baloh, MD;
`Michel D. Ferrari, MD, PhD;Joanna C.]en, MD, PhD;
`Am M.]. M. van den Maagdenberg, PhD
`
`102
`
`~-Synuclein Gene Rearrangements
`in Dominantly Inherited Parkinsonism:
`Frequency, Phenotype, and Mechanisms
`Pablo Ibanez, PhD; Suzanne Lesage, PhD; Sabine Janin, BS;
`Ebba Lohmann, MD; Frank Durif, MD; Alain Destee, MD;
`Anne-Marie Bonnet, MD; Christine Brefel-Courbon, MD;
`Simon Heath, PhD; Diana Zelenika, PhD; Yves Agid, MD, PhD;
`Alexandra Durr, MD, PhD; Alexis Brice, MD;
`for The French Parkinson's Disease Genetics Study Group
`
`ARCH NEUROL/VOL 66 (NO. 1), JAN 2009
`7
`
`WWW. ARCHNEUROL.COM
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`

`

`ARCHIVES
`NEUROLOGY
`
`109
`
`BOOK REVIEW
`
`Functional Neuroimaging in Clinical Populations
`Eduard Kraft, MD
`
`137
`
`CORRESPONDENCE: RESEARCH lETIER
`
`Longitudinal Cortical Atrophy in Amyotrophic
`Lateral Sclerosis With Frontotemporal Dementia
`Brian Avants, PhD; Alea Khan; Leo McCluskey, MD, MBE;
`Lauren Elman, MD; Murray Grossman, MD, EdD
`
`138
`
`116
`
`CORRESPONDENCE: COMMENTS AND OPINIONS
`
`Motor Nerve Hyperexcitability and
`Muscle Cramps in Machado-Joseph Disease
`Kazuahi Kanai, MD, PhD; Satoshi Kuwabara, MD, PhD
`
`121
`
`In Reply
`Marcondes C. Frani;a]r, MD; Anelyssa D'Abreu, MD;
`Anamarli Nucci, MD, PhD; Iscia Lopes-Cendes, MD, PhD
`
`A Case of Neuromyelitis Optica With
`Gadolinium-Enhancing Braitt lesions
`and Parinaud Syndrome
`Christopher P. Gilmore, MRCP;
`Anujacob, MD; Nihos Evangelou, MRCP
`
`In Reply
`Man Gotkine, MBBS
`
`REGULAR DEPARTMENTS
`
`Online CME Quiz Questions
`
`Annual Reviewers list
`
`This Month in ATchives of Neurology
`
`Announcements
`
`125
`
`130
`
`Diffusion Tensor Imaging in Sporadic
`and Familial (D90A5OD1) Forms
`of Amyotrophic lateral Sclerosis
`Biba R. Stanton, MRCP; Daisy Shinhrnar, BSc;
`Martin R. Turner, PhD; Victoria C. Williams, MRCP;
`Steven C. R. Williams, PhD; Camilla R. V. Blain, MRCP;
`Vincent P. Giampietro, PhD; Marco Catani, MRCPsych;
`P. Nigel Leigh, FMedSci; Peter M. Andersen, DMSc;
`Andrew Simmons, PhD
`
`OBSERVATIONS
`
`Continued Disease Activity in a Patient
`With Multiple Sclerosis After Allogencic
`Hematopoietic Cell Transplantation
`]ian-Qiang Lu, MD, PhD;
`Jan Starek, MD, PhD; Luanne Metz, MD;
`V. Wee Yong, PhD; Anne M. Stevens, MD, PhD;
`Richard A. Nash, MD;Jeffrey T.]oseph, MD, PhD
`
`Amyotrophic lateral Sclerosis-Plus Syndrome
`With TAR DNA-Binding Protein-43 Pathology
`Leo F. McCluskey, MD, MBE;
`Lauren B. Elm.an, MD; Maria Martinez-Lage, MD;
`Vivianna Van Deer/in, MD, PhD; Wuxing Yuan, MS;
`Dana Clay, CCC; Andrew Siderowf, MD;
`John Q. Trojanowski, MD, PhD
`
`Alzheimer Abnormalities of the Amygdala
`With Kliiver-Bucy Syndrome Symptoms:
`An Amygdaloid Variant of Alzheimer Disease
`Shawn]. Kile, MD; WiUiam G. Ellis, MD;
`Jolin M. Olichney, MD; Sarah Farias, PhD;
`Charles DeCarli, MD
`
`IMAGES IN NEUROLOGY
`
`Primary Cerebral Whipple Disease
`Presenting as Kluver-Bucy Syndrome
`Wolfgang Leesch, MD;
`Ingeborg Fischer, MD; Robert Staudinger, MD;
`Douglas C. Miller, MD, PhD; S-wati Sathe, MD
`
`139
`
`139
`
`140
`
`140
`
`11
`
`15
`
`17
`
`20, 43, 67, 72, 78,
`84, 101,129,141
`
`39
`
`91
`
`14 3
`
`143
`
`Diffusion-Weighted Magnetic Resonance Imaging
`o( Bilateral Simultaneous Optic Nerve Infarctions
`Joshua P. Klein, MD, PhD; Adam B. Cohen, MD;
`W. Taylor Kimberly, MD, PhD; Anhoor S. Shah, MD, PhD;
`Yannek I. Leidennan, MD, PhD; Dean M. Cestari, MD;
`Marc]. Dinkin, MD
`
`132
`
`Online CME Article
`
`Call for Papers
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`Classified Advertising
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`Journal Advertiser Index
`
`Giant Cell Tumor of the Sphenoid
`Maria Margarita Company, MD; Rafael Ramos, MD
`
`134
`
`Instructions for Authors
`www.archneurol.com/instructions
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`ARCH NEUROL/VOL 66 (NO. l), JAN 2009
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`A Renaissance for Antisense Oligonucleotide
`Drugs in Neurology
`Exon Skipping Breaks New Ground
`
`Toshifumi Yokota, PhD; Shin'ichi Takeda, MD, PhD; Qi-Long Lu, MD, PhD;
`Terence A. Partridge, PhD; Akinori Nakamura, MD, PhD; Eric P. Hoffman, PhD
`
`A ntisense oligonucleotides are short nucleic acid sequences designed for use as small(cid:173)
`
`molecule drugs. They recognize and bind to specific messenger RNA (mRNA) or pre(cid:173)
`mRNA sequences to create small double-stranded regions of the target mRNA that
`alter mRNA splicing patterns or inhibit protein translation. Antisense approaches have
`been actively pursued as a form of molecular medicine for more than 20 years, but only one has
`been translated to a marketed drug (intraocular human immunodeficiency virus treatment). Two
`recent advances foreshadow a change in clinical applications of antisense strategies. First is the
`development of synthetic DNA analogues that show outstanding stability and sequence specificity
`yet little or no binding to modulator proteins. Second is the publication of impressive preclinical
`and clinical data using antisense in an exon-skipping strategy to increase dystrophin production
`in Duchenne muscular dystrophy. As long-standing barriers are successfully circumvented, atten(cid:173)
`tion turns toward scale-up of production, long-term toxicity studies, and the challenges to tradi(cid:173)
`tional drug regulatory attitudes presented by tightly targeted sequence-specific drugs.
`Arch Neural. 2009;66(1):32-38
`
`With the advent of recombinant DNA in
`the 1970s, it was soon realized that bac(cid:173)
`teria possess a form of regulatory machin(cid:173)
`ery where small RNA transcripts can bind
`(hybridize) to other target RNAs and in(cid:173)
`hibit the translation of these targets. 1 These
`anti sense R As were subsequently recog(cid:173)
`nized as natural translational regulation
`mechanisms in plants and higher organ(cid:173)
`isms.2 More recently, a specialized form of
`antisense transcript was found to be a cel(cid:173)
`lular defense mechanism against invad(cid:173)
`ing messenger R As (mRNAs) (viruses),
`and this has been harnessed as a popular
`method to "knock down" specific mRNA
`transcripts in cultured cell models (short
`interfering RNAs) .3
`Attention soon shifted toward devel-
`opment of antisense molecules as a form
`
`Author Affiliations: Research Center for Genetic Medicine, Children's National
`Medical Center, Washington, DC (Ors Yokota, Partridge, and Hoffman);
`Department of Molecular Medicine, National Institutes for Neuroscience, Tokyo,
`Japan (Drs Takeda and Nakamura) ; and McColl-Lockwood Laboratory for
`Muscular Dystrophy Research, euromuscular/ALS Center, Carolinas Medical
`Center, Charlotte, North Carolina (Dr Lu).
`
`of small-molecule drug (antisense oligo(cid:173)
`nucleotide [AO]). The approach was in(cid:173)
`tuitive: one needs simply to chemically
`synthesize short pieces of D A of about
`20 bases, where a specific complemen(cid:173)
`tary sequence is designed to hybridize with
`a desired target mRNA. Such designer AO
`drugs should show very high specificity
`and selectivity for binding only the de(cid:173)
`sired target RNA sequence of nucl~otides
`that is predicted by base pairing. Begin(cid:173)
`ning in the mid-1980s, this approach was
`put to the test in model systems and was
`shown to work quite well in shutting down
`the production of the target ( undesired)
`protein ... Isis Pharmaceuticals, Inc, Carls(cid:173)
`bad, California, a company focused on
`clinical applications of AOs, was incorpo(cid:173)
`rated in 1989. Additional companies fo(cid:173)
`cusing on AO approaches soon followed.
`Despite early promise, uses of AOs as
`small-molecule drugs have been pain(cid:173)
`fully slow to enter the market and stan(cid:173)
`dard of care. Indeed, only a single AO drug
`has been approved by the Food and Drug
`
`ARCH NEUROL/ VOL 66 (NO. 1), JAN 2009
`32
`
`WWW.ARCHNEUROL.COM
`
`

`

`ONA
`
`RNA
`
`2'-0-MeAO
`
`H
`AcHN-(RXR),X / N ~ o
`
`Base
`
`___,..,ease
`
`(_~
`I
`/
`O=P- N
`I
`"-
`
`o f-o Base
`
`N:)J I
`
`0
`
`O=P -
`
`0
`
`I -
`~
`
`LNA
`
`PMOs
`
`PPMOs
`
`Figure 1. Comparison of chemistries used for the exon-skipping approach. Examples of artificially developed antisense oligomers such as 2' -0-methylated
`antisense oligonucleotides (2'·0-MeAO) (phosphorothioate), locked nucleic acid (LNA), phosphorodiamidate morpholino oligomers (PMOs), and peptide-tagged
`PMOs ( PPMOs) are shown for comparison with DNA and RNA.
`
`Administration (FDA), fomivirsen sodium (Vitravene; Isis
`Pharmaceuticals, Inc) delivered by intravitreous injec(cid:173)
`tion to inhibit cyt.omegalovirus retinitis in AIDS. Vit(cid:173)
`ravene was approved in 1998 and there have been no sub(cid:173)
`sequent successful approvals in the ensuing 10 years.
`What has slowed the progress of AO drugs into the
`clinical arena, and why may this be changing?
`There have been 2 major hurdles: off-target toxic ef(cid:173)
`fects and potency or delivery. Regarding toxic effects, most
`organisms do not take kindly to covert infiltration by for(cid:173)
`eign DNA or RNA. Indeed, all species have quite effec(cid:173)
`tive mechanisms to destroy foreign DNA and RNA as they
`are more likely than not to be viruses or other undesir(cid:173)
`able organisms. In addition, many of the clinical trials
`testing AO drugs have seen evidence of activation of the
`complement cascade, and this has been a key concern of
`the FDA. Delivery has also been a consistent problem.
`Because the target RN As are always intracellular, it is im(cid:173)
`perative for the AO drug to achieve intracellular concen(cid:173)
`trations sufficient to enable it to bind and modulate the
`target RNA to a significant extent. The fact that A Os typi(cid:173)
`cally do not easily cross the lipid bilayers that bound the
`
`cell so as to achieve sufficient intracellular potency via
`systemic (intravenous) delivery has been problematic.
`Recent developments are achieving success in over(cid:173)
`coming both hurdles. Analogues of nucleic acid have
`been designed and synthesized in which the ribose
`backbone of RNA and DNA is replaced with different
`chemistries (Figure 1 ). Two are particularly promis(cid:173)
`ing: one uses a morpholino backbone (phosphorodi(cid:173)
`amidate morpholino oligomer [PMO]; AVI BioPhanna,
`Portland, Oregon) , and the second uses a locked
`nucleic acid backbone (Enzon Pharmaceuticals, Inc,
`Bridgewater, New Jersey). These new backbones are
`designed to maintain the molecular distan ce between
`bases (G, A, TIU, and C), enabling highly sequence(cid:173)
`specific base pairing to the target RNA that is stronger
`in the case of PMO and locked nucleic acid drugs than
`DNA or RNA AOs. Equally important, these backbones
`are so dissimilar from the DNA and RNA ribose phos(cid:173)
`phodiester backbone that they are not recognized by
`most or any DNA and RNA binding proteins or degrad(cid:173)
`ing enzymes, thereby enhancing their stability and
`avoiding many or all off-target toxic effects.
`
`ARCH NEUROUVOL 66 (NO. 1) , JAN 2009
`33
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`WWW.ARCHNEUROL.COM
`
`

`

`The second major barrier has been achieving sufficient
`intracellular concentrations (delivery). One successful ap(cid:173)
`proach is to take advantage of preexisting holes in the plasma
`membrane of the target cell. Infecting viruses breach the
`cell membrane during the process of infection and appear
`to bring along AO drugs in the process. As such, A Os have
`been quite successful in blocking downstream viral repli(cid:173)
`cation within cells, and PMO drugs are showing impres(cid:173)
`sive promise as antiviral antidotes.5 Another preexisting hole
`is found in muscle cells lacking dystrophin (Ducheru1e mus(cid:173)
`cular dystrophy [DMD]).6 The unstable plasma mem(cid:173)
`brane of myollbers app ears to allow the AO to leak into
`the cell. 7 An additional approach is to modify the AO drugs
`with cell delivery moieties, chemical adducts that pen(cid:173)
`etrate the cell membrane. One example is the addition of
`arginine-rich peptides to one end of the AO drug (peptide(cid:173)
`tagged PMO) (Figure 1).
`From these advances has sprung a resurgence of in (cid:173)
`terest in AO drugs for treatment of genetic disease, can(cid:173)
`cer, and infectious disease. The purpose of most appli(cid:173)
`cations is to knock down a target RNA so that it makes
`less of the deleterious protein product (eg, tumor growth
`factor 13 or hypoxia-inducible factor la in cancer cells,
`viral mRNAs, or dominant gain-of-function toxic pro(cid:173)
`teins in inherited neurological disease). However, the dis(cid:173)
`order that may be most advanced in such applications is
`DMD. Here the AOs are used for a quite different objec(cid:173)
`tive than for previous applications; explicitly, A Os in DMD
`are designed to restore function to the target mRNA and
`protein rather than b lock it. The remainder of this re(cid:173)
`view focuses on this application.
`
`RATIONALE AND PROOF OF PRINCIPLE
`OF EXON-SKIPPING THERAPY
`
`The principle of exon-skipping therapy for dystrophi(cid:173)
`nopathies was initially demonstrated by Dunckley et al8
`in cultured mouse muscle cells in vitro. The rationale is
`as follows . Duchenne muscular dystrophy is caused by
`mutations of the 79-exon gene (commonly deletions of
`;;::1 exon). Within the myofiber, the remainder of the gene
`will be transcribed and spliced together. However, if the
`triplet codon reading frame of the mRNA is not pre(cid:173)
`served, the resulting frame shift will lead to the failure
`of dystrophin protein production. Becker muscular dys(cid:173)
`trophy (BMD) is a clinically milder and more vatiable dis(cid:173)
`ease in which mutations of the dystrophin gene are com(cid:173)
`monly such as to preserve the translational open reading
`frame; thus, after splicing together, the remainder of the
`gene retains some ability to synthesize the dystrophin pro(cid:173)
`tein. The goal of exon-skipping therapies is to force the
`dysfunctional mRNA with out-of-frame mutations in a
`patient with DMD to skip (exclude) some additional ex(cid:173)
`ons. The loss of additional matetial directed by AO drugs
`restores the reading frame, changing a Duchenne out(cid:173)
`of-frame transcript to .a Becker in-frame transcript. For(cid:173)
`tunately, most mutations in the dystrophin gene occur
`in parts that do not code for functionally essential re(cid:173)
`gions of the protein.
`This AO-mediated exon-skipping method has been de(cid:173)
`veloped and extensively tested on the dystrophic mdx
`mouse model of DMD. The mdx mouse harbors a non-
`
`sense mutation in exon 23 that prevents translation be(cid:173)
`yond this point in the transcrip t. Both local intramuscu(cid:173)
`lar injection and systemic delive1y of a single AO targeted
`against exon 23 in the primary transcript excludes this
`exon from the mRNA, leaving an in-frame transcript that
`generates dystrophin expression and produces a degree
`of functional recovery. Intramuscular and systemic in(cid:173)
`jections of A Os for exon splicing of a dog model of DMD
`have also been demonstrated with a novel cocktail AO
`strategy (T.Y., S.T., Q.-L.L., T.A.P.,A.N., E.P.H., and Ma(cid:173)
`sanori Kobay ashi, DVM, unpublished data, 2006-
`2008) . The principle is similarly illustrated in humans;
`van Deutekom et al9 reported single-site intramuscular
`injections of 2' -0 -methyl AO chemistry in 4 boys with
`DMD, showing evidence of de novo dystrophin produc(cid:173)
`tion at the injection site.
`These data demonstrate that the key hurdles of achiev(cid:173)
`ing intracellular delivery and avoiding toxic effects can
`be cleared. A similar strategy is being explored in other
`diseases such as myotonia, human immunodeficiency vi(cid:173)
`12
`rus, and spinal muscular atrophy. 10
`·
`
`HURDLES IN BRINGING EXON SKIPPING
`TO STANDARD OF CARE
`
`Exon skipping using AO drugs has rapidly emerged as the
`frontline therapeutic approach foi: DMD. How soon can we
`expect exon skipping to reach the neuromuscular clime and
`standard of care? This approach is breaking new ground
`and raising challenges not encountered previously in drug
`development. Different patients have different mutations,
`and many AO sequences will need to be designed, tested,
`and FDA approved. Also, current genotype and pheno(cid:173)
`type data suggest that there may be good in-frame dele(cid:173)
`tions and not-so-good in-frame deletions; simply restor(cid:173)
`ing the reading frame may not be synonymous with
`resto1ing dystrophin protein function. The optimization of
`dystrophin function will likely require deletions of mul(cid:173)
`tiple exons, and tl1is will require mixtures of different AOs(cid:173)
`new territoty for drug development and the FDA. The ap(cid:173)
`proach will require regular injections of large amounts of
`AO drug; what are the long-term toxic effects? Moreover,
`are the standard toxicity tests appropriate for sequence(cid:173)
`specific drugs? Each of these hurdles is discussed briefly
`in the remainder of this review.
`
`CERTAIN EXON DELETIONS MAY RETAIN MORE
`DYSTROPHIN FUNCTION THAN OTHERS
`
`The molecular diagnostics of DMD and BMD frequ ently
`refer to the reading frame rule, where out-of-frame de(cid:173)
`letions are given a DMD diagnosis and in-frame dele(cid:173)
`tions are given a BMD diagnosis. However, as many as
`30% of patients with BMD do n ot adhere to this rule. 13 A
`thorough understanding of reading frames is critical for
`appropriate design of exon-skipping therapies, both so
`that the best AO can be given to the patient and so that
`an optimally functional dystrophin protein is produced
`as a result of the expected exon skipping. Currently, the
`best information from which to predict the capabilities
`ofpartiaily deleted dystrophins to rescue the DMD phe(cid:173)
`notype comes from analysis of the thousands of geno-
`
`ARCH N EUROL/VOL 66 (NO. 1) , JAN 2009
`34
`
`WWW.ARCHNEUROLCOM
`
`

`

`[Kl
`
`100
`
`80
`
`"#-
`ci
`:::;; 60
`0
`.c
`~
`,,,
`40
`~
`~
`20
`
`0
`
`45
`
`47
`
`48
`49
`Deletion Start Exon
`
`50
`
`[ID
`
`100
`
`80
`
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`0
`£
`~
`.l/l 40
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`
`46
`
`47
`
`48
`
`49
`Deletion End Exon
`
`51
`
`53
`
`55
`
`Figure 2. Clinical phenotypes associated with specific start (A) and end (B) sites for in-frame deletions. Percentages of patients with Duchenne muscular
`dystrophy (OMO) out of patients with DMD or Becker muscular dystrophy with specific start and end exons are shown. Combined muscular dystrophy databases
`of 14 countries (from Argentina. Belgium, Brazil, Bulgaria, Canada, China, Denmark, France, India, Italy, Japan, The Netherlands, the United Kingdom, and the
`United States) at Leiden University (http://www.dmd.nl), where diagnoses were perforimed using multiplex ligation-dependent probe amplification/multiplex ampli·
`fication and probe hybridization, Southern blotting. or polymerase chain reaction primer sets that allow deletion boundaries to be assigned accurately to a specific
`exon, are used (deletion start sites: n =288 for exon 45, n=23 for exon 47, n=9 for exon 48. n=12 for exon 49, and n =1 O for exon 50; deletion end sites: n= 11 for
`exon 46, n= 115 for exon 47, n=95 for exon 48, n=51 for exon 49, n=53 for exon 51, n=40 for exon 53, and n=21 for exon 55).
`
`type and phenotype correlations in patients with DMD
`and BMD that have been published in the literature and
`on the Internet. We examined all in-frame deletions and
`determined the proportion of observed cases that showed
`mild or severe phenotypes. This was gleaned fro m com(cid:173)
`bined muscular dystrophy databases of 14 countries (from
`Argentina, Belgium, Brazil, Bulgaria, Canada, China, Den(cid:173)
`mark, France, India, Italy, Japan, The Netherlands, the
`United Kingdom, and the United States) at Leiden Uni(cid:173)
`versity (http://www.dmd.nl), excluding diagnoses that did
`not allow deletion boundaries to be assigned accurately
`to a specific exon. 14 Of all observed in-frame deletion pat(cid:173)
`terns on genomic DNA in the central rod domain hot(cid:173)
`spot region (exons 42-57; 28 distinct patterns), 57% (16
`of 28 patterns) were associated with DMD rather than
`BMD. This analysis showed that there are considerable
`discrepancies betwten population-based ratios and pat(cid:173)
`tern-based proportions of severe DMD vs mild BMD phe(cid:173)
`notypes, and interestingly, the ratio of DMD to BMD re(cid:173)
`markabl