Neuromuscular disorders . Nn
`·
`DUP - General Collection
`W1 NE337GB
`v. 20, no . 2
`Feb. 2010
`
`20 (2) 95- 154
`ISSN 0960-8966
`
`Neuromuscular
`Disorders
`
`Editor-in-Chief
`V Dubowitz UK
`Associ ate Editors
`AG Engel USA
`N laing Australia
`L Merlini Italy
`1 Nishino Japan
`A Oldfors Sweden
`T Voit France
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`PROPERTY OF THE
`NATIONAL
`LIBRARY OF
`MEDICINE
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`Official Journal of the World Muscle Society
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`Neuromuscular Disorders
`Editor-in-Chief
`V Dubowitz
`(See ac/cl,-ess below)
`Editorial Mana er
`J Miller, The Dllbowitz NeL1romL1scu /a,- Cent,-e, Jsr Floo,-, UCL lnsritute
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`Aims and Scope
`This internal iona l, multidisciplinary journ,1I covers all aspects or 11euro111u,cu la1 cli , 01 cle1 s in chilclhoocl .incl aclulI lil l' ( inclucli11g I IIL' mu,nil.ir cly,I ropllil•,. ,pi11,1I
`muscular aIrophies. herecl it<1ry neuropa1hies, congenit,1I rnyopa thi es, 111yastheni,1,. 111yotonic syndromes, 111ct,1bol ic 111yop,11hic, ,111(1 i11l l,1111111.1I01y 111yop,1Il11l's).
`The Edi tors welcome origina l articles from all areas of 1he fi eld :
`• Clinical aspects, such as new clinical enl ities. case studi es of interest, trea t 111en1. management and reh,1bil it,1 t ion ( i nclucli 11g bio111cch,111ic,, 0 111101 it dt•sig11 ,IIHI
`surgery):
`• Basic scientific studies of relevance to the clinical synd ro mes, including advances in the fields or molecular biology and gc11cI ic,:
`• Studies of animal models relevant to the human diseases.
`The journal is aimed at a wide range of clinician s, pathologists. associated paramedical proressionals and clinical and basic ,de11I1,I, wi l h ,111 i11Ic1t•,I 111 Jill'
`study of neuromuscular clisorclers.
`In addition to original research papers: 1he journal also publishes reviews and mini- reviews. prelimi nary shon co1nmu11ic,11 iom and book reviews, ,111cl h,1,
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`are also welcome.
`The journal is published twelve times a year and ai ms al rJ pid publication of high-qu,1lity papers of scie111ific merit ,incl general i11Iere,I 10 ,1 wick I e,1clc1 sh ip.
`There is also a fast track for rapid publica1io11 of new material of outsl ancling scientific merit and im port ance.
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`Printed by Polestar Wheatons I.tel, Exeter, UI<
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`(D
`Neuromuscular
`Disorders
`
`Editor-in-Chief
`V Dubowitz
`
`Volume 20 No. 2 (2010)
`
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`Neuromuscular Disord ers 20 (20 I 0) I 02- 1 I 0
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`Contents lists available at ScienceDirect
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`Neuromuscular Disorders
`
`ELSEVIER
`
`journal homepage : www. e ls e vier .com/ locate / nmd
`
`Comparative analysis of antisense oligonucleotide sequences targeting exon 53
`of the human DMD gene: Implications for future clinical trials
`Linda j. Popplewell a. ,, Carl Adkin b. 1, Virginia Arechavala-Gomeza b, Annemieke Aartsma -Rus c,
`Christa L. de Winter \ Steve D. Wilton d, Jennifer E. Morgan b, Francesco Muntoni b, Ian R. Graham a,
`George Dickson a .•
`
`•1 Sc/100/ of FJiologirnl Scie11ces, /loyal Holloway - University of t.011do11, Eglw111 , Surrey TW20 0EX, u,,ired Ki11gdo111
`" Dubowirz Neuro11111srn lar Ce111re, Uct lrrsriru1e of Child Hec1l//1, 30 Guilford Sr reel. London WCI N I El I, Uuired Ki11gdo111
`'Ceur erfor l·l11mw1 and Clinirn l Generics, Leiden U11iversiry Medical Ceur er, PO FJox 9600, 23001/C Leiden, Tire Ne1/rerlc111c/s
`dCenrre for Neurolog ical and Neuromuscu lar Disorders, Ausrra lia11 Neuromusrn lar llesea rc/1 /nsriwr e, U11iversiry of Wesren, Aus1ralia, Perr Ir, WA 6009, Aus1ra lia
`
`ARTICLE
`
`INFO
`
`A B S T R A C T
`
`Arricle l1isro1y:
`Received 13 July 2009
`Rece ived in revised form 20 October 2009
`Accepted 27 October 2009
`
`Keywords:
`Exon skipping
`Duchenne muscular dystrophy
`Anti sense oligonucleotides
`l'hosphorodiamidate morpholino ol igomer
`
`Duchenne muscular dystrophy (DMD ) is ca used by the lack of fun ctional dystrophin protein, most com(cid:173)
`monly as a res ult of a ran ge of out-of- fram e mutations in the OMO gene. Modulation of pre-mRNA splic(cid:173)
`ing with anti sense oligonu cleotid es (AOs ) to restore the reading frame ha s been demon strated in vitro
`and in vivo, such that truncated but functiona l dystrophin is ex pressed. AO- induced skipping of exon
`5 1 of the OMO gene, w hich could trea t 13% of DMD pati ents, has now progressed to clini cal trials. We
`describe here the methodical, cooperative compari so n, in vitro (in DMD ce ll s) and in vivo (in a tran sge ni c
`mou se ex pressing human dystrophin ), of 24 AOs of the phosphorodiamidate morpholino oli gomer ( PMO )
`chemi stry designed to targe t exo n 53 of the OMO gene, skipp ing of whi ch cou ld be potentially app li cab le
`to 8% of patients. A number of the PM Os tested should be considered worthy of develop ment for clinica l
`trial.
`
`([) 2009 Elsevi er B.V. All rights reserved.
`
`1. Introduction
`
`Duchenne muscular dystrophy (DMD ) is a ,eve, ' musclr wast
`ing disease, affecting I :3500 live mal r bi,th s, ca used by th e l,1ck of
`functional dystrophin protr in in skrlrtal mu scl 'S, ,is a result of
`frame-di srupting de let ions or clupli ca t ion s or, lrss common ly, non
`sense or missense mutati ons in th e OM/J gene I 11. MuI,111ons th ,1 1
`maintain the rea ding fram e or th r grnr and ,1 llow expre,,ion of
`semi - fun ctional, but intern ally-delr ted dy, trophin are gener,1lly
`associated with
`the
`lrss srvrrr Brcker muscular dystrophy
`(BMD) 11 ,2 1.
`Tran sforming an out-of- framr DMD mutation into its in- fram e
`BMD counterpa rt with anti se nse oligonucleot ides (AOs) is th e ba sis
`of th e potentially exciting exon skipping th erapy for DMD (re(cid:173)
`vi ewed by Muntoni and W ells) 131. Th e hybridiza ti on of AOs to spe(cid:173)
`cific RNA sequence motifs prevents assemb ly of the spliceosome,
`so that it is unabl e to recogni se th e target exon(s) in the pre-mRNA
`and include them in the mature gene transcript 14,5 ]. AOs have
`been used to induce skipping of specific exons such that the rea d(cid:173)
`ing fram e is restored and trun cated dystrophin expressed in vitro
`
`Corresponding au th or. Tel. : +44 (0) 1784 443870: fax: +44 (0) 1784 4 14224.
`E-mail address: g.dickson@rhul.,1c.uk (G. Dickson ).
`' Th ese aut hors contribut ed equally 10 1his paper.
`
`0960-8966/$ · see front 1na11 er " 2009 Elsevier 13.V. All rigl1ts reserved.
`doi : I 0. 101 6/j .nrncl.2009. 10.0 13
`
`in DMD patient cells 16,5,7- 9 ], and in anima l models of the disease
`in vivo 14,10- 131.
`Initial proof-of-principle clinica l trial s, using two different AO
`ch ·mistri es ( phosphorothioate-linked 2'-O-methyl modified bases
`(2'OMe l>S) I I4I and phosp horod iam idate morpholino oligomer
`( PM O) 11 51) for I hr targeted skipping of exon 51 of th e OMO gene
`,1lI er intr,1 muscul ar injection, have been perform ed recrntly with
`enco u,aging r suits. Whi le both chemi stri es hav exce llent safety
`profilrs I 16, 17 I, J>MOs appear to producr more co ns is ten I and sus-
`1,1incd rxo n skipping in th r mclx mouse modr l of I MD I I 8- 201, in
`humdn mu scle rx plant, 12 1 I, and dy, trophi c canine musclr cr ll s
`in vi tro 1221. I lowever, for so me human •xo ns, 2'OMrPS and
`PM O AON s perform ·cl equally wr ll I 171,
`ince th e mutations that
`ca usr DMD arr so clivr rse, or th ose DMD pa1i 0 n1 s with ge nomic
`dr l tion s, skipping or rxo n 5 1 wo uld h,,v, 1hr pot ential 10 Irr,1 1
`only 13% of such patient s on th e Lr id n DMD cl at,1b,1se 12'3 1. ,incl
`I r:: % of such patients on th r UM D DMD Fr,rnce mul,1t ion~ clat,1b,1se
`(see http://www.umd .b /D MD/4A l'I ON/W MONO ). Althou gh any
`predi ction s on the frequ ency of mutati ons and pr rcr ntag or skip(cid:173)
`pabl e pati ents should be viewed w ith ca ution , it is und eni.:tbl r that
`the continued development and analysis of AO · for th e targeting of
`other DMD exo ns is vital.
`Here we report th e compara ti ve ana ly is of,, seri es of PM Os tar(cid:173)
`geted to exon 53, skipping of whi ch wou ld have th e potential to
`
`

`

`/ .. }. l'opplewe/1 et ol./ Ne11ro11111srn lor Disorders 20 (20 10) 102 I 10
`
`103
`
`treat a furth er 8% of DMD pati ents with geno111ic deletions on th e
`Leid en databa se 123 1. and a furth er 13.5% of pati ents on the
`UMD-DMD Fran ce 111utations data base (see http: // www.umcl.be/
`DMD/4ACrJON /W _MONO ). PMOs des igned and previou sly te tecl
`in normal human skel etal mu scle cell s (hSkMCs) for th e targeting
`of exo n 53 I 24 I were furth er studi ed here in cell s from a DMD pa(cid:173)
`tient with a releva nt del etion (clel 45- 52). Th ese PMOs were di(cid:173)
`rectly compared to a PMO based on a AO previou sly identifi ed as
`being th e most bioa ctive by Wilton et al. I25 I. Time-course studi es
`w ere perform ed to evaluate the persiste nce of skipping and close(cid:173)
`res ponses were exa mined. Findings from these experim ents were
`supported by in vivo studi es in a mouse model tran sge ni c for th e
`entire human clystrophin locus 11 21. Col lectively, thi s work reports
`a nu111ber of PMOs ab le to produ ce targeted skipping of exon 53 to
`levels that would suggest them worthy of consideration for
`upcoming PMO clini ca l trial s.
`
`2. Materials and methods
`
`2. I. AO design
`
`All AOs were synthesized as phosphoroclia111idate morpholino
`oligomers (PMOs) by Gene Tool s LLC (Philomath OR, USA).
`
`2.2. DMD patient primmy myoblast culture
`
`Skeleta l musc le biopsy samples w ere taken from a diagnostic
`biopsy of the quadriceps from a DMD patient with a del etion of
`exo ns 45- 52. Informed co nse nt was obtained before any process(cid:173)
`ing of samples, and all work was ca rri ed out with the approval of
`the in stitutional ethi cs committee. Muscle precursor cell s were
`prepared from the biopsy sample by sharp di ssection into I mm 3
`pieces and di saggrega ted in solution conta ining HEPES (7.2 mg/
`1111 ), NaCl (7.6 mg/ 111I ), KCI (0.224 111g/111l ), glucose (2 111g/ml ), Ph e(cid:173)
`nol reel ( 1.1 pg/ml ), 0.05% Trypsin- 0.02% EDTA (lnvi trogen, Pai sley,
`UK ) in cli still ecl water, three times at 37 °( for 15 111in in Whea ton
`nasks w ith vi gorou s stirring. Iso lated ce ll s w ere plated in non(cid:173)
`coa ted plastic nasks and cu ltured in Skeleta l Mu scle Growth Media
`(Promocell, Heidelberg, Germany) suppl emented with 10% Foetal
`Bovine Serum (PAA Laboratories, Yeovil, UK), 4 111M L-glutamine
`and 5 pg/111I genta111ycin (S igma - Aldrich, Poole, UK ) at 37 °C in
`5% CO2.
`
`2.3. Nucleof ection of OMO prima,y myoblasts
`
`Betwee n 2 x 105 and 1 x 106 ce ll s/ ml w ere pelleted and resus(cid:173)
`pend ed in I 00 pl of so luti on V (Amaxa Biosystems, Cologne, Ger(cid:173)
`many ). The appropriate PMO to skip exon 53 was added to the
`cuvette provid ed, sufficient to give the concentrations desc ribed,
`followed by th e ce ll suspension, and nucleofected using the Ama xa
`nucleo fector 2, program B32. Five hundred microliters of mecliu111
`was aclclecl to th e rnvette i111111ed iately following nucleofection
`126 1. Thi s suspension was transferred to a 6 well plate in differen(cid:173)
`liJtion m edium. Nucleofected cell s were 111aintained in differenti (cid:173)
`ation media for 3- 21 days post treatm ent before extraction of
`RNA or protein. Transfections were performed blindly and in each
`ex periment in tripli cate. Each experiment was repea ted at leas t
`once to ensure reproducibility of res ults.
`
`2.4. Lc1c1c11e dehydrogenase cyrotoxicity assay
`
`A sampl e of medium was taken 24 h post-tran sfection to assess
`cy totoxicity by release of lactate clehyclrogenase (LOH ) into the
`111eclium , using th e LOH Cytotoxicity Detection Kit (Roche, Burgess
`Hill , UK), fo ll owing the manufacturer's in structions. The mean of
`
`three rea dings for each samp le was reco rd ed, with medium only,
`untreated and dead con trol s. The readings were normali sed for
`backgro und (minu s medium onl y) and percentage toxicity ex(cid:173)
`pressed as l(sa mpl e-untrea tecl )/( cleacl-untrea tecl ) x lOOJ.
`
`2.5. Transgenic lwman OMO mice
`
`A tran sge nic mou se ex press ing a compl ete copy of th e human
`OMO gene has bee n generated I 12,27 ]. Experiments were per(cid:173)
`formed at the Leiden Uni ve rsity Medical Ce nter, with the authori (cid:173)
`zation of the Animal Experim ental Commi ss ion (UDEC) of the
`M edi ca l Farnlty of Leid en University as described prev iously I 9 J.
`Twenty mi crograms of eac h PMO was inj ected once in to two gas(cid:173)
`trocnemius mu sc les, pretreated with ca rdiotoxin. Mice were sacri (cid:173)
`fi ced 1 week after the injection, and RNA harvested from the
`iso lated gastrocnemiu s muscles and analysed by RT- PCR.
`
`2.6. /?NA isolation and reverse tmnscriplion -polymemse chain reaaion
`c11wlysis
`
`RNA wa s iso lated and ana lysed by RT- PCR, as desc ribed previ (cid:173)
`ously 19]. Primer seq uences and detail ed PCR protoco ls are avai l(cid:173)
`ab le on request. PCR products were analysed on 1.5% (w/v)
`agarose gels in Tris- borate/EDTA buffer. Skipping efficiencies were
`determined from gel images by co mparing indu ced shortened dys(cid:173)
`trophin mRNAs to the intact transcript of the full length using den(cid:173)
`sitometric analysis with Image J so ftware (for pati ent sa mpl es) or
`by quantifying the skipped products w ith ONA 1000 LabChip Kit
`on th e Agilent 2100 bioanalyze r (Agil ent Techn ologies, USA) ( for
`hDMD mouse samples). Skipping perce ntages were ca lcu lated as
`th e amount of skip tran sc ripts relative to the total tran sc ripts (s kip
`and full length ). Equa l amounts of the indu ced and intact tran(cid:173)
`scripts would be rega rd ed as represe nting 50% effici ency, w hil e
`an estimate of 25% exo n skipping wou ld be represented by the in (cid:173)
`ta ct tran script being three times more abundant t han the band
`represe nting th e indu ced transcript. Likewise, if the induced tran(cid:173)
`script was prese nt at three times the level of the intact transcript,
`the exo n skipping effi ciency would be assessed to be 75%. Where
`appropriate, the two-tailed student's I-test was used to determine
`the stati sti cal strength of th e sk ipping effici encies produced.
`
`2. 7. Sequence analysis
`
`RT- PCR products were exc ised from agarose gels and extra cted
`with a QIAqu ick gel extraction kit (Qiagen, Crawley, UK). Direct
`DNA sequ encing wa s ca rri ed out by the MRC Genomics Co re
`Facility.
`
`2.8. Western blot analysis of dyslrop/Jin protein
`
`DMD pati ent cell s, transfected as described and cultured in dif(cid:173)
`ferentiation medium, were harvested 7, 14 or 2 1 clay s post-tran s(cid:173)
`fection. Cell s (4 x 105) were pell eted and res uspend ed in 50 pl or
`load ing buffer (75 mM Tri s- HCI pH 6.8, 15% sodium clodecyl sul(cid:173)
`phate, 5% ls-mercaptoethano l, 2% glycero l, 0. 5% bromoph enol blu e
`and comp lete mini protease inhibitor tabl et). Sa mpl es were in cu(cid:173)
`bated at 95 °C for 5 min and centrifuged at I 8,000g for 5 min.
`Twenty microliters of sampl e wa s loaded per w ell in a 6% poly(cid:173)
`acrylamide gel with 4% stac king gel. Protein from CHQ5B ce ll s dif(cid:173)
`ferentiated for 7 clays was used as a positive co ntrol for clystrophin.
`Gels were electrophoresecl for 5 hat 100 V before blotting on nitro(cid:173)
`cellul ose membrane at 200 mA overnight on ice. Blots w ere stained
`w ith protogolcl to assess protein loading, th en blocked in 10% non (cid:173)
`fat milk in PBS with 2% Tween (PBST) for 3 h. Blots were prob ed
`with antibod ies to dystrophin, NCL- DYSl (Vector Labs, Peterbor(cid:173)
`ough, UK) diluted 1 :40 and to dysferlin, Hamletl (Vector Labs)
`
`

`

`104
`
`LJ. Popplewell er ol./ Net1ro111usculor Disorders 20 (2010) 102- 110
`
`diluted 1 :300 in 3% non-fat milk/PBST. An anti -mou se, biotinylated
`secondary antibody (diluted 1 :2000; GE Healthcare, Amersham,
`UI<) and streptaviclin/horse radi sh peroxidi se conjugated antibody
`( 1: 10,000; Dako, Ely, UI<) allowed vi sualisa tion in a luminol-HRP
`chemiluminescence reaction (ECL- Plus; GE Healthca re) on Hyper(cid:173)
`film (GE Healthcare), exposed at intervals from IO s to 4 min.
`
`2.9. Statistirnl analysis
`
`For the blind comparison at 300 nM in DMD pati ent cells, data
`from two separate experiments performed in duplicate and tripli (cid:173)
`cate respectively were pool ed and compared by two-tai led student
`t-test. Dose- response and time-course experiments were com(cid:173)
`pared by two-tail d, paired 1-tcst.
`
`3. Results
`
`Twenty-four AOs clesignecl to target exon 53 of the OMO gene
`have been previou sly tested in normal human skeletal mu sc le ce lls
`(hSkMCs) 124,25). Table 1 summarises the names and targe t se(cid:173)
`quence characteristics of these AOs (shown in bold ), and % skipping
`produced by each in normal hSkMCs. However, studies in normal
`hSkMCs are limited as th ey clo not allow assessment of the thera(cid:173)
`peutic effect at the protein level (i.e. clystrophin restoration ). Fur(cid:173)
`ther studies have therefore been performed here to elucidate and
`confirm which AO(s) would have th e potential as a trea tment for
`pati ents with an eligible deletion. AOs, whose target sites are with (cid:173)
`in the sequence +29 to + 74 of exon 53 , th e region previously shown
`to be in open confo rmati on, binding to whi ch interferes with
`spli ceosome-mecliated pre-mRNA spli cing, such that exon 53 is
`skipped 124,25 I, were directly compared in exon 53 -skippable pa(cid:173)
`tient ce lls (at UCL), and in the humanised DMD (hDMD ) mouse (at
`LUMC). The AOs w ere all synthesized as PMOs to all ow direct com(cid:173)
`pari son or skipping effi cacy. While PMOs w ere hybridi zed to
`mi xed-backbone DNA leashes in the previous study I 24), t he nucle(cid:173)
`ofection method used here wa s performed on unleashed PMOs.
`
`3. 1. Comparison of PMOs 10 exon 53 in ()Ml) pcrcient cells
`
`Our compa rative eva luation of l'M O inclucecl ex on skipping effi
`ciencies wa s perform ed in a blinded f,1 shion. /\ II t1a11 ~fcctio11 s w ere
`performed in tripli ca te and repe,1tecl Jl lec1 st on ce to emu 1c unifor
`mity of results. Skipping effi ciencies were determined from In PCR
`gel images by comparing induced shortened clystroph1n ml~N/\s to
`the intact full length transcript using clcn ~itometril ,111.ilysis, as cle
`scribed previou sly 125 I. Seq uencing of RT-l'CR products confi1 med
`the targeted skipping of exon 53 (result s not shown ). For quJntifi(cid:173)
`ca tion , th e skip- products were a11Jlysecl using densitometric ,rna l(cid:173)
`ys is witl1 Image J so ftware. Thi s technique for qua ntifyi ng skipping
`effi ciencies of AOs targeted l o the DMD gene has been publi shed
`previously /9, l 7/. Rea l-lime PCR quantification of int.let and in(cid:173)
`clucecl transcripts ha s proven to be imposs ible clue to a number
`of obstacles (variation of ampli fication effi ciencies of each tran (cid:173)
`script, possible interference of intact and induced transcript prim (cid:173)
`ers/probes with each other) (results not shown). No DMD exon
`skipping studies thus far reported have included rea l-tim e PCR
`quantification of AO efficacy, and we believe we have used the best
`method avai lable for quantification. Skipping effi cie ncy is given as
`th e percentage of skip transcript over th e total amount of tran(cid:173)
`script (skip and fu ll length). AOs were sub-divided on the basis
`of their skipping efficiency. PMOs that produced over 50% exon
`skipping w ere designated as Type I , those that produced between
`25% and 50% exon skipping w ere described as Type 2, while those
`that produced less than 25% as Type 3. Where appropriate, the
`
`two-tail ed student's t-tes t was used to assess significa nt differ(cid:173)
`ences between AOs.
`The 13 PMOs, whose target sites are within the sequence +29 to
`+ 74 of exon 53, were com pa reel directly at a 300 nM close by nucle(cid:173)
`ofection [26 1. Thi s dose was se lected for comparison, since such
`concentrations of AOs have been used in numerous previous exon
`skipping studies in DMD 15,6,91. PMOs-G, - Hand -A w ere th e most
`efficient, producing a mea n of 73% (±4.10%), 68% (±4.77%) and 68%
`(±4.14%) exon skipping respectively ( cla ss ified as Type I ) (Fi g. I ).
`The other PMOs tested prod