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`Act;i nl1/0iogic;a : m;·opatbie$ a!i:! cardiomyo(lt!:)
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`(Myopathies, Cardiomyopathies and Neuromyopathies) ,
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`Official Journal of the Mediterranean Society of Myology
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`Four-monthly
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`

`

`CONTENTS
`(Papers published in Acta Myologica are available in pdf on our website www.cardiomiologia.it)
`
`CONTENTS
`
`Cover letter
`
`ORIGINAL. ARTICLES
`
`What do animal models have to tell us regarding Duchenne Muscular Dystrophy? Del Wells and
`KE Wells ....
`
`Stem cells to treat Muscular Dystrophies. JE. Morgan
`Gene tlwrapy for Ducl1enne muscular dystrophy: AAV leads the way LM. Judge, JS. Chamberlain
`0/igonucleoticie-medintecf gene editing for neuromuscular disorders. C Bertoni
`Non-Vim! /\µproaches For Geno Transfer J. Wolff, David L. Lewis, Hans Herweijer, Julia Hegge and
`. . . . . . . . . . . . . . . . . . . . . . . . . .
`James H,1gstrorn
`Utrop/1i11 u1ireuulatio11 in Ouci1cn110 Muscular Dystropliy RC . Hirst, KJA McCullagh and l<E Davies .
`The Mocfulntion of Skclotnl Muscle Glycosylntion as a Potential Therapeutic Intervention in Muscular
`Dystrophios M BrockinDton mid F Mu11toni
`/\ntiscnso oliuonuclooticfos. axon skipping and the dystrophin gone transcript. S D Wilton and
`S Flutcilcr
`Molcculnr Mechanisms involving IGF- I ancf Myostatin lo Induce Muscle Hypertropl,y as a therapeutic
`..... .. .. ...... .....
`strategy for Duchenno Musculnr Oystroµliy I< Patel, R Macharia, H Amthor .....
`
`NEWS TROM AROUND THE WORl.D
`...... . . . .
`.. .
`MSM
`
`GC/\
`
`WMS
`
`Summer sc/100I of myology
`[uro/JiolH111k
`
`FORTHCOMING MEETINGS
`/w;/rtl(:tions for /\u/lwrs
`
`169
`
`172
`
`181
`
`184
`
`194
`
`202
`
`209
`
`2 17
`
`222
`
`230
`
`242
`
`242
`
`242
`
`242
`
`242
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Acta Myo/ogica • 2005; XX/V p. 222-229
`
`An11:isense oligonucleotides, exon skipping
`and the dystrophin gene transcript
`
`S. 0. WILTON AND S. FLETCHER
`Experi111e11tal Molernlar Medicine Group, Centre for Nc11ro11111srn/ar and Neum/ogi('(I/ Disordt'rs
`Unil'l'l"sity <f Westem J\ustmlia
`
`Antisense oliJlotlllcfeolide induced exon skipping has rl'l'ently
`emerJled as a potential thernpy to hy-pass the consL•11uem·cs of'
`many, hut not all dystrophin mutations that lead to Ducl1enne
`muscular dystrophy. Targeted remoml of one or more exons,
`lo restore a disrupted reading frame, or omit a nonsense mula(cid:173)
`tion, eould lessen the consequences ofan estimatrd 80% ofdys(cid:173)
`trophin gene mutations. l'romising iu 1·itm and iu l'il'II exper(cid:173)
`iments in m1imal models of dyslro(lhino(lalhies, as well as
`demonstrntion of indm:cd exon ski(lpin1-: in l'ullun·d human
`myogenic cells have prompted rnnsiderahle enthusiasm. Fur(cid:173)
`thermore, advances in anliscnsc oligormclcolide chcmislrics
`ha,•e resulted in the de\'l'lopmcnt of more stable and less tox(cid:173)
`ic compomuls, some of which arc currently in Phase 111 clini(cid:173)
`cal trials for selected antiviral applkalions. This review will
`summarize devcln(llllenls in in1h1ccd c:rnn skippin1-: tfml hm·c
`paved the way to clinical trials and some of the d1:tllrn1-:es and
`flOSsihle limitations.
`
`Keywords: Al1ern:11ive spiking, f{evcr1a111 Fibres, M111atio11
`suppression. D11che1111c muscular dystrophy
`
`Introduction
`Mutations in the dystrophin gene that preclude
`the synthesis of a functional protein lead to Duchenne
`muscular dystrophy. In developing a treatment for
`DMD, compensating for the defective dystrophin
`gene has now been recognised as a much greater
`challenge than originally anticipated. Potential ther(cid:173)
`apies have included cell (my~iblast. satellit~ and st~m
`cell) or gene replacement (viral and non-viral deliv(cid:173)
`ery) (for review sec 11.21, read-through of nonsense
`mutations 13.41, corticosteroids 15-81 or inhibition
`or sped fie prnteoly_sis 19, IO I '.ind the s~tbject or this
`review, antiscnse oltgonucleottde (AO) induced exon
`skipping. Of all these approaches. it is only the lat(cid:173)
`ter that has any natural precedent.
`Dystrophin positive revertant fibres 111] in dys(cid:173)
`trophic tissue arise from _an unknm~n exon skipping
`mechanism 112-141. wl11le the variable phenotypes
`
`observed in Becker muscular dystrophy patients clear(cid:173)
`ly demonstrate that some in-frame, inteinal deletions
`of dystrophin, particularly in the rod domain, can
`result in a protein of near normal function 115-17 ].
`Furthermore, although chemically synthesised
`antisense oligonucleotide (AO) analogues cannot be
`regarded as natural compounds. small, naturally
`occurring, non-coding RNAs have been identified and
`implicated in the control or a variety of cellular
`processes 118 !. Small RNAs have been shown to
`silence selected genes 1191 and modify gene cxpn:s(cid:173)
`sion at the level or splicing or trnnslation I 201. There(cid:173)
`fore. the application orAOs to modify gene transcripts
`for therapeutic outcomes should not be regarded as
`whimsical.
`
`Na111ml /Jl'l'l'('dt'l//.1'.fr1r (//1 Hro11 Ski11pi11g (IJIJJroOch
`to addJ"l'.\'S dystmp/1i11 111111a1io11s
`Rcvertant fibres were reported in the 11u/.r mouse
`1111 and in DMD p:1tie11ts 1211 and so named because
`or 'reversion' to the normal dystrophin staining pat(cid:173)
`tern . Various dystrophin 111RNA transcripts excluding
`the primary genetic lesion, a11d in which the reading
`frame has been restored or maintained have been
`described in hu111an, canine and murine dystrophic
`tissue 112, 22, 231- It is now clear that revertant fibres
`result from an exon skipping mechanism, and that not
`all have the same cxonic combination I 131- /11 sit11
`hybridi1.ation studies using a dystrophin intron 21-
`exon 25 genomic probe on mdx mouse muscle showed
`that the dystrophin gene was intact in the majority or
`revertant fibres , and RT-PCR and antibody epitope
`mapping indicated that the most com111011 exon skip(cid:173)
`ping rearrangements involved 20 or more exo11s 11 JI.
`With the apparent exclusion or secondary somat(cid:173)
`ic genomic deletions within the dystrophin gene
`being the cause of rcvertant fibres . the n1echa11is111
`
`/\ci~fr~;;· r;;·· corrcsµo11clonco. S.D. _Wilton. S Flotchor. Centro for Nouromuscular ii/IC/ Nourotouical Di:;orclur:;, Nt>c lla11<1:;_ /\/ 1:;(cid:173)
`tmlia. University of Western li ustralra. Emmi: sw1//011(ii}cyllo110. uwD.ociu.i111 . . sf/o/c;f,(<»cyllonu.uwn.m/11 nu
`
`This m.ateria1222.ied
`at the N LM and may be
`Seubj ect US,Copyright Laws
`
`

`

`Antisense oligonucleotides, exon skipping and the dystrophin gene transcript
`
`responsible for generating dystrophin is most likely
`to involve a localized alteration in splicing. Since
`revertant fibres occur singly or in small clusters, sug(cid:173)
`gesting a clonal origin I 11.13, 24], the events that
`bring about exon skipping must only occur within the
`dystrophin-positive fibres and not in the surrounding
`muscle. Small non-coding RNAs have recently been
`credited with controlling aspects of gene expression,
`from splicing to translation [ 19,20]. The possibility
`exists that the revertant fibres express novel microR(cid:173)
`NA variants that interfere with dystrophin pre(cid:173)
`mRNA processing.
`Apart from conlinning the existence and utility
`of exon skipping in the dystrophin gene transcript.
`another important property of revertant librcs is that
`they not only demonstrate immune tolerance to dys(cid:173)
`trophin. but 111ay also play a causative role in the
`development of this tolerance 125.2(1 [. Depending
`upon the nature and position or the mutation, produc(cid:173)
`tion or amino terminal frag111cnls and dystrophin iso(cid:173)
`forllls from internal pro111otcrs would also expose
`the illllllllne systc111 to various dystrophin epitopes.
`Consequently, an ill1111unc response to any induced
`dystrophin in individuals who have rcvcrt,111t fibres
`is considered unlikely. although the possibility or
`novel epitopes encoded hy the induced exon junc(cid:173)
`tions cannot he excluded.
`The dystrophin gene rcarrange111cnts in mildly
`affected BMD patients clearly de111011strate that so111c
`dolllains arc 1101 essential t'or 11ear-11or111al function.
`The reading fra111c rule 1271 holds true for the 111ajor(cid:173)
`ity of dystrophin mutations. Nonsense or frame shift(cid:173)
`ing 111utations result in pre111ature termination or
`translation and the absence or a functional protein
`leads to DMD while in-frame deletions cause BMD
`1271. In some cases. the consequences or a dclction
`arc so mild that the individual is asymptomatic and
`may only be diagnosed later in life 11 (1, 17.28,291.
`There appears to he an upper limit to the sit'c of in(cid:173)
`frame deletions that lllay be tolerated. where the loss
`or J4 or more cxons is invariably associated with a
`severe phenotype !JOI.
`
`f.ff<'f>lions lo lh<' /'{'{11/ing .fiWII<' !1_1·110//,<'sis OJ/(/
`th<' ll<'<'<i for 1m·ci.\'I' 11111tatio11 d1·t1•1 ·tio11
`Apart from rare llliS-Sl'llse 11111tations in crncial
`binding donwins of the dystrophin gene. n1;111y or
`the apparent exceptions to the reading frame rule
`may he l'Xplained whl'n till· l'L'SIH111sihle sl'condary
`n1echanis111s arl' idl'ntilil'd. So11Jl' dystrophi11 11011-
`Sl'llSl' 111111atio11s do tllll lead to l)J'vll), sillL'L' thL' base
`
`change compromises motifs involved in pre-mRNA
`processing lJ I ,J21. In these cases, the nonsense
`mutation may prevent efficient exon recognition hy
`the splicing machinery and the exon is variably
`excluded from the mature dystrophin mRNA. If loss
`of the exon docs not disrupt the reading frame, the
`nonsense mutation is removed from the mature dys(cid:173)
`trophin gene transcript and a slightly shorter, BMD(cid:173)
`like protein can be produced. The amount of func(cid:173)
`tional dystrophin generated, and hence the severity
`of the phenotype, reflects the degree to which the
`exon is excluded 1321. If the effect of the base change
`were to marginally weaken splicing, generating only
`a small percentage of the transcripts missing the
`mutation, a more severe phenotype would be predict(cid:173)
`ed. Conversely, if exon skipping as a result of the
`nonsense mutation was complete and assuming that
`the lost coding domain was not essential, the patient
`could be asympto111atic.
`It has been esti111ated that so111e 15%, of hu111an
`mutations alter splicing IJJ !. Changes in primary
`splicing motifs that may be readily identified include,
`the branch-point, acceptor and donor splice sites.
`Other DNJ\ changes that may alter splicing can be
`less obvious. particularly when a single base change
`deep within an intrnn results in the inclusion of a
`pseudo exon 1341. lntronic changes over 10 kilobas(cid:173)
`es from the nearest coding sequence have been
`shown to alter the processing of dystrophin exons
`I 35 [. Exonic splicing enhancers, motifs recognised
`by splicing factors such as the SR-proteins can be
`predicted in silico IJ(il but accurate identification
`occurs when a particular exonic base change modi(cid:173)
`fies the splicing pattern 1371. J\n apparently neutral
`polymorphism (C>T change al the third base of
`codon 608 in the lamin A/C gene) is responsible for
`Hutchinson-Gilford Progeria Syndrome 138-401.
`This d,, nm ·o substitution activates a cryptic splice
`site 5 bases upstrca111 that leads to the loss of 150
`nucleotides fro111 the gene transcript 1401.
`It is cxa111plcs such as these that emphasize the
`need for detailed molecular characterization in dis(cid:173)
`ease diagnosis. so that 1101 only arc DNJ\ changes
`detected. 11111 the consequences or the alterations are
`considered. Fmthennore. precise 111t1tation detcc1io11
`will be essential prior to the application of targeted
`thernpies such as splicing manipulation. The bound(cid:173)
`aries of the genomic deletions or duplications must
`he clearly dclined so that the appropriate target site
`can he characterized for the design of J\Os to restore
`thl' 1"L·ading frame. Similarly. any exon carrying a
`
`Thism~ efi:?was copie. cl
`at the 'Ftulvl .a,nd may be
`'>u:bject US Copyright Law s
`
`

`

`S.D. Wilton et al
`
`nonsense mutation must be identified and a strategy
`developed to remove that exon, and flanking cxons
`if necessary to maintain the reading frame. Many
`exons in the dystrophin gene, particularly those
`encoding the rod domain, would not disrupt the read(cid:173)
`ing frame if excluded. Excision of a single exon
`should be sufficient to overcome a majority of the
`prematurely truncating mutations. It may also be nec(cid:173)
`essary to confirm the sequence of the AO annealing
`site to eliminate the possibility of natural DNA vari(cid:173)
`ation compromising AO annealing.
`
`uncharged backbone and exhibit remarkable biolog(cid:173)
`ical stability. as there are 110 enzymes that can recog(cid:173)
`nize and degrade the morpholine structure 146).
`Metabolic breakdown products of PM Os have not yet
`been detected and the uncharged backbone does not
`appear to exhibit non-antisense effects, as do AOs on
`PS backbones. Several PM Os are under evaluation as
`antiviral agents with promising results, and a PMO
`has entered clinical trials 1521, http://www.antivi(cid:173)
`rals.com/devNeugcne.html and no overt adverse
`effects reported lo date.
`In application to both splicing and translation
`suppression, AOs must be carefully designed to
`the susceptible target motifs and should preferably
`be resistant lo degradation. An ;\() with a brief
`biological half-life may only affccl splicing or
`translation for a limilcd period and changes in
`expression may 1101 be readily detectable. Biolog(cid:173)
`ically stable AOs would exert a more sus1aincd
`effect, depending upon the hall'-lil'e of the gene
`product and the turnover or the tissue. Improve(cid:173)
`ments in the design and synthesis of AOs, have
`yielded novel che111istries, including PMO which
`show 110 overt adverse effects 153-55 l and persist
`and exert biological activily for extended periods
`I 56 J. The major isoforms transcribed from the dys(cid:173)
`lrophin gene arc relatively stable and expressed in
`cells wilh slow turnover, and ii is not unrealistic
`lo expect that periodic administration of /\Os cou Id
`induce and 111ai11lai11 therapeutic benelils.
`Another factor to be considered is the extent 10
`which gene expression must be altered to obtain
`the desired benefit. In targeting a viral or oncogene
`transcript for suppression, the degree of gene down(cid:173)
`regulation may need lo be irbsoltllc lo achieve ther(cid:173)
`apeutic benefit. Conversely, in order lo aller the
`course of a disease such ilS DMD. only ii propor(cid:173)
`tion of gene lrnnscripls may need to be 111odified.
`Defective dyslrnphin gene lranscripts cannot encode
`a functional prolcin and arc likely lo be subjected
`to nonsense mediated decay, while any modified
`in-frame transcripts would undergo repeated rounds
`of translation and allow the protein to gradually
`accumulate with time.
`
`AO modification of splicing
`Richard Kole (Chapel Hill. North Carolina) pio(cid:173)
`neered the application or AOs to address splicing
`mutation in the f3-globin gene 157-591. These
`researchers used AOs to mask cryptic splice sites in
`lhc introns or lhc Ii-glob in gene lranscripls 10 no1111al-
`
`Mechanics of Antisense O/igonucleotide
`111aniJJulatio11 <f gene e.rJJression
`AOs were originally applied to down-regulate
`specific gene expression, most commonly through
`the induction of RNascH, an enzyme that degrades
`the RNA strand of an RNA:oligodcoxyribonu(cid:173)
`cleotide duplex. Unmodified oligonuclcotidcs have a
`natural phosphodicster backbone that renders these
`compounds susceptible to nuclease degradation. One
`chemical modification to increase oligonuclcotidc
`stability is the substitution of the non-bridging oxy(cid:173)
`gen atom of the backbone with sulphur 141 I. The
`resultant phospl10rothioatc (PS) backbone confers
`greater resistance to degradation, but concomitantly
`elicits some non-anti sense effects I 42 I.
`Additional increases in stability were achieved
`by modifying the ribosc moiety of the oligonu(cid:173)
`clcotide. Advantages of the 2'-O-mcthyl (2OMc)
`modified bases on a PS backbone include greater
`resistance to nuclease degradation, but perhaps
`even more significantly, these compounds do not
`induce RNaseH activity when annealed to an !{NA
`target [43,44 ]. Instead of targeting an mRNA tran(cid:173)
`script for RNascH degradation, appropriately tar(cid:173)
`geted 2OMeAOs bind to motifs in the primary
`gene transcript and may interfere with cxon recog(cid:173)
`nition and/or spliccosomc assembly 145 J.
`It is possible, therefore lo alter gene expression
`through a variety of mechanisms. In addition to
`RNaseH degradation of a target gene transcript, gene
`down-regulation may be brought about by AOs
`directed to motif-; involved in translation. Suppres(cid:173)
`sion oftranslation by AOs with novel backbone
`chemistry, phosphoro-amidc morpholino oligonu(cid:173)
`cleotidcs (PMOs) 146.47). has been very effective in
`a number or systems /48-50]. PMOs directed
`upstream of the initiation codon or the target mRNA
`arc able to prevent ribosome binding and subsequent
`protein translation /51 J. These compounds have an
`
`Th is2~ I w as CO'pied
`
`at the NLM and may t>e
`S.ubject US Co,pyright Laws
`
`

`

`Antisense oligonucleotides, exon skipping and the dystrophin gene transcript
`
`izc the splicing patterns. This concept has been re(cid:173)
`interpreted to address DMD. Normal splice sites of
`selected cxons arc targeted to induce abnormal dys(cid:173)
`trophin prc-mRNA processing to bypass mutations,
`which would otherwise prematurely terminate trans(cid:173)
`lation.
`The first report of AO-induced cxon skipping in
`the dystrophin gene transcript was from the laborato(cid:173)
`ry of Professor Masafumi Matsuo, (Kobe Japan), who
`used an oligodcoxyribonuclcotide (ODN) of 31 bases
`directed at a motif in human dystrophin exon 19 l60J.
`Exon 19 skipping was induced in human cultured
`lymphoblastoid cells, although oligonuclcotidcs of
`this chemistry arc more commonly used to induce
`degradation of target mRNJ\. A likely explanation for
`this is that once the RNJ\/ODN duplex was excised
`from the transcript. along with the flanking sequence,
`the resultant mf{NA would 110 longer be susceptible
`lo RNascl I degradation.
`J\ variety of J\O chemistries have now been eval(cid:173)
`uated to modify dystrophin cxon processing and the
`most co111111only used to date arc the 2OMcAOs I (JI -
`(J7 I. Recently. we reported that 2OMcJ\Os were
`much more effective at inducing dystrophin cxon
`skipping than ODNs of identical sequence in i11 l'i!(cid:173)
`m studies 1681. l1 is now apparent that PMOs arc
`even more crticicnl than the equivalent 2OMcAOs
`when administered by intramuscular injections in the
`md.r 1nouse model of muscular dystrophy I 5(J ].
`
`/Jesig11 rf 110s for i11d11c('(/ e.ron skiJJJJiflg
`Obvious targets for J\O in1crven1ion arc !he accep(cid:173)
`tor and donor splice silcs. l111racxo11ic sites involved
`in cxon rccog11i1io11 and splicing arc called cxonic
`splicing enhancers (ESEs) and these can he predict(cid:173)
`ed to some cxlenl i11 silim j:l(J]. Erringlon ct al /69]
`undertook a study of splicing nwlifs within, and flank(cid:173)
`ing cxon 19 thal could he largcled lo induce skipping
`of that cxrn1. Exon 19 was found to lie :cadily omit(cid:173)
`led from the mature 1111111;111 dystrophin gene lranscript
`after :1pplicatio11 of J\Os directed al !he acceptor and
`donor splice sites, as well as shorter J\Os targeting
`IIH>lifs within 11Jc JI nucleotide domain idcn1ilicd by
`f>ra11101w cl al. j(iOI as containing an !:SI:. J\11 ;\() of
`only I ,f n11clco1idcs was able lo i11d11cc L'.\CIJ1sio11 of
`L'Xon 11), all>eil only al"tcrad111i11islra1ion al rclaliwly
`high rn11ce11tra1io11 /<> 1)1.
`Once a s11i1ahk 111<>lif !hat r.111 lie 111ash·d lo dis(cid:173)
`nip! splil-eoso111L' assen1iily and i11d11rl' ro11sis1L·11I and
`s11sl:1ined largl'l l'rnn skippin_e has lil'l'll idl'nlificd.
`lo fmlhn
`additional options iil'ro111c a,·ailalill'
`
`enhan~e the biological effect. These include tenni(cid:173)
`nal modifications 1681, other chemistries [70-73 land
`agents to enhance uptake f65,74J.
`Judith van Deutekom, and collegues f2,73 ,75-
`78J have undertaken extensive studies on sup(cid:173)
`pressing many DMD mutations. In addition they
`have also developed a "humanized transgenic
`mouse" which carries the entire human dystrophin
`gene 1791. Although the human dystrophin cxons
`are recognised and correctly processed by the
`murinc splicing machinery, an important outcome
`of this work was the demonstration of the exquisite
`sensitivity of AOs in targeting specific sites. AOs
`directed at the human gene transcript could modify
`splicing of that transcript, but still have no effect
`011 the corrcspo11ding region of the murinc dys(cid:173)
`trophin mRNA. Although AOs may be regarded
`as exerting comparable specificity to that of a PCR
`primer, the potential of an AO to cross-react with
`a homologous but unrelated mRNA cannot be dis(cid:173)
`counted. However, this may not prove to be of
`any great concern. It has been our experience in
`designing AOs for dystrophin cxon skipping that
`moving an oligonuelcotide target site by only a
`few nucleotides can alter the biological effect by
`an order of magnitude (unpublished observations).
`
`Clinical trials
`J\11 ENMC workshop was held in Naarden. the
`Nclhcrlands to discuss clinical trials using AOs in
`DMD 1801. At the time, the most advanced plans
`were presented by the Dutch/Belgian group. who
`propose to exclude cxon 51 as the initial target. This
`was to be undertaken with a 2OMc AO on a phospho(cid:173)
`rolhioatc backbone directed at an cxon 51 ESE. The
`therapeutic compound will be injected. in the absence
`of delivery enhancing agent, as a single dose in sev(cid:173)
`er.ii injections in the ti/Jialis m1tl'J'io1: A biopsy will
`be taken 28 days later for analysis of RNA and pro(cid:173)
`tein. Patient recruitment is cu1-rcn1ly ongoing and the
`!rials arc cxpeclcd to he completed by dming
`:200(J.Trials in the United Ki11gdo111 would be of a
`similar na111rc, bu! with so111c variation lo avoid direct
`d11plic:11ion. Regardless of the line details of the J\O
`lo he cn1ployed or the muscle lo he lrcatcd. it was
`decided thal there should he consislL'ncy and cross(cid:173)
`validalion of the results obtained in the studies.
`i11cl11di11g AOs, arc generally
`Nucleic ,1cids.
`not efficiently taken up by cells, parlicularly in in
`1·itm s111diL'S. A 11111nhn of agents arc available
`11Ja1 c,111 facili1:1te intra cellular delivery of J\Os.
`
`1')' '
`
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`

`S.D. Wilton et al
`
`including polycthylcniminc, cationic liposomcs
`and block copolymers. It is important to note that
`not all of these agents perform similarly in vitro
`and in vivo. Lipofectin or Lipofcctaminc 2000 can
`deliver charged AOs into cultured cells or muscle
`with, but at a cost of moderate cell damage. The
`block copolymer Fl 27 has been used to introduce
`20MeAOs in vivo f 65,66], however, this agent is
`ineffective in vitro.
`The different AO chemistries will have specific
`requirements for cell delivery and nuclear uptake.
`The delivery, and subsequent exon skipping efficien(cid:173)
`cy, of 20McAOs is substantially improved by the use
`of agents to enhance delivery and uptake [ 63-65]. We
`recently reported that PMOs can be administered in
`vivo without a delivery agent, a feature that should
`facilitate their use in clinical trials. Data using a PMO
`to by-pass the dystrophin cxon 23 mutation in the
`mdx mouse have indicated this chemistry to be supe(cid:173)
`rior to the 20McAOs [561. Under parallel condi(cid:173)
`tions, uncomplcxcd AOs were administered to mice
`by intramuscular injection and only the PMOs were
`able to induce persistent dystrophin expression. In
`contrast, the equivalent 20McAO was unable to gen(cid:173)
`erate any correctly localized dystrophin as deter(cid:173)
`mined by immunohistochemical staining or western
`blotting. The 20McAO, when administered at high(cid:173)
`er concentrations did induce some cxon skipping,
`however, at these dosages, the AOs arc more likely
`to cause adverse or non-specific effects.
`
`The Future
`The use of AOs to alter gene expression by redi(cid:173)
`recting splicing should not be regarded as a 'gene
`therapy'. Although synthetic nucleic acid analogues
`can manipulate the processing of the prc-mRNA. the
`DNA remains unaltered and no permanent genetic
`changes arc introduced. Once the AO has been
`removed from the system, the cxon skipping effect
`will be lost and any benefit derived from the treat(cid:173)
`ment will only persist while the modified gene tran(cid:173)
`scripts and the induced, internally shortened dys(cid:173)
`trophin protein remains in the cell. It has become
`clear from several studies that dystrophin is a partic(cid:173)
`ularly stable protein and. since muscle fibres have a
`slow turnover, AO induced dystrophin may be cor(cid:173)
`rectly localized for weeks or months after a single
`treatment [ 56,66.(17 [.
`Ncvcrthclcss, if cxon skipping docs prove to be a
`viable therapy. periodic administration ofAOs will be
`essential to maintain clinically beneficial levels of
`
`dystrophin. The frequency of AO re-administration
`required will depend upon a number of variables,
`including the route of administration, efficiency of
`uptake as well as persistence and duration of AO
`action. Based upon current animal studies using PMO
`preparations, we postulate that a weekly injection reg(cid:173)
`imen of between 5 and 25 mg PMO per kg will be
`required to induce detectable dystrophin that would
`have the potential to ameliorate the severity of DMD.
`The prolonged half-life of this protein and the slow
`turnover of muscle fibres would suggest that a main(cid:173)
`tenance dose would then be necessary, possibly at
`less frequent intervals. Potential delivery regimens
`include intravenous and subcutaneous delivery, how(cid:173)
`ever investigation on modes of further enhancing and
`improving systemic delivery is ongoing.
`Repeated administration of AOs to maintain
`dystrophin expression is not without risk. No infor(cid:173)
`mation on the consequences of long-term admin(cid:173)
`istration of PMOs or other antisense chemistries
`to humans is currently available. Although PMOs
`appear to be the chemistry of choice for the ini(cid:173)
`tial AO DMD trials, it is imperative that other
`chemistries continue to be evaluated, in the event
`that there arc unanticipated adverse effects asso(cid:173)
`ciated with these compounds.
`Exon skipping can be induced by natural RNA.
`using adeno-associatcd viral constructs containing
`expression cassettes coding for antiscnsc RNA
`sequences under the control of U7 promoters. Goy(cid:173)
`anvillc ct al., I 81 [ clearly demonstrated the utility of
`this approach in the 111dr mouse. This work has rccrnt(cid:173)
`ly been successfully extended to the canine golden
`retriever muscular dystrophy model. Although
`extremely promising, viral AO delivery must be
`regarded as a gene therapy and confront the chal(cid:173)
`lenges associated with the use of viral vectors, such
`as achieving sustained expression, overcoming
`immunological reaction and high production cost.
`
`Co11c/11di11g co111111e11ts
`Luis Garcia (Gcncthon, France) has referred to
`the cxon skipping strategy as an "a la carte" therapy.
`Unique compounds will be required to address the
`numerous mutations occurring across the dystrophin
`gene, but this may not prove to be an onerous task
`because of the existence of hot-spots for genomic
`deletions. It has been proposed that a small number
`of AOs would address the majority of these dys(cid:173)
`trophin deletions [76[. Nonsense mutations occur at
`an estimated frequency of I 5<1<• of DMD cases and
`
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`

`Antisense oligonucleotides, exon skipping and the dystrophin gene transcript
`
`appear to be distributed evenly across the dystrophin
`gene. This will necessitate a panel of AOs that will
`allow removal of any dystrophin exon that does not
`code for a crucial functional domain, since the rod
`domain consists of almost half the coding sequence.
`The Perth laboratory has developed AOs to target
`over 50 exons in the dystrophin gene transcript.
`Although some exons remain to be addressed, and
`certain AOs require further optimisation, a number
`of potentially therapeutic sequences arc now avail(cid:173)
`able for application in clinical trials.
`Despite the enthusiasm and anticipation for the
`upcoming AO clinical trials, the expectations must be
`realistic. Exon skipping cannot "cure" DMD. At best,
`cxon skipping may reduce the severity, with the
`extent of any benefit greatly influenced by many fac(cid:173)
`tors, including the age of the patient, the nature and
`position of the mutation, as well as the ef'liciency and
`persistence of the AO effect. However, if AO thera(cid:173)
`py docs induce synthesis of some functional dys(cid:173)
`lrophin in patients, a substantial delay in the pro(cid:173)
`gression of this disorder would be anticipated. The
`outcome of the AO therapy may be further improved
`if other treatments shown lo be of benefit, including
`coticostcroids, arc also included.
`
`Acknowledgements
`The authors would like to acknowledge sup(cid:173)
`port from National Medical & Health Research
`Council of Australia. National Institute of Health
`USA, Muscular Dystrophy Association of USA,
`aktion bcnni and co and Parent Project Muscular
`Dystrophy USA.
`
`References
`I. Flelchcr S, \Villon SD. and I lowcll JM (