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`vol, 17 no. 3 nrnrch 2009
`wivw, 11wlcc11/a rthcrnpy,org
`
`Biocnginccrcd skin:
`working the hugs out
`Exploiting millNJ\s for vector engineering
`Neural stem cells target brain metastases
`
`MIEIUCAN
`SOCIET\'of
`_ &, GENE
`.
`Th 1s matenal w ascop i~ THERAPY
`atth-e NLM and m a y be
`5'uot;ject US Co:pyright Laws
`
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`namrc p11hli.~hi11g group I_· •_' ;-4
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`

`

`Molecular
`______ Th era RY-_w_1_1•1_1•._11_u_,,_ct_·11_1i_,r_l_h_e,_·11_p_y._o_rg ______________________________ _
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`oprnrons appeMing in the ,Hliclt•s ,rnd ,1dvrrli1,•n1<'nl1 1,,-r,•in ,Hr \h t• tl'lfl<lll ·
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`Society of Gene Thcr,1py, 111<• p11t,lii her1 ,rnd the l'ri1tor1 .rnd tll<'ir "''l"'l liv,·
`employees, officPrs ,Hid ,HJe11t1 ,1cc rpl no li,11,ility wh.1l101•v1•r Im 1111• l om,•(cid:173)
`quenc cs of .iny rnch in,1tc11r,lh· or mill,•,1<li11q d .11,1. 11pir111111 or 1t.11,·111,•11I.
`
`Th i:s m.ate ria I wa:s co-pied
`at the N LNI a n.d may be
`5,ubj-ect USCop,yright Laws
`
`

`

`contents
`
`Molecular
`Therapy vol.17 no. 3 march 2009 - - - - - - - - - - - - - - - - - - - -
`editorial
`The RAC: Double, Double, Toil, and Trouble?
`H Ertl
`
`397
`
`On tl,e cover:
`Inhibition of multidrug-resistant
`Acinetobacter baumannii by non(cid:173)
`viral expression of hCAP-18 in a
`bioengineered human skin tissue .
`See the article by Thomas-Virnig
`et al. on pages 562-569.
`
`400
`
`401
`
`403
`
`405
`
`409
`
`417
`
`425
`
`4 30
`
`439
`
`448
`
`455
`
`463
`
`4 72
`
`in this issue
`research highlights
`co111nzentaries
`AAV9: A Potential Blood-Brain Barrier Buster
`Ff' Manfrcd1rnn, AC Rising and Rf Mandel
`Bioengineered Human Skin: Working the Bugs Out
`L 5tei111tm<'11cr, 5 Al-/lcnna, M Kc1ti11g 011d F /acobsc11
`review
`VECTOR ENGINEERING AND DELIVERY
`MicroRNAs and the Regulation of Vector Tropism
`[f Kelly 011d 5/ R1111cll
`originnl nrticles
`MONOGENIC DISEASE
`Enhanced Factor VIII Heavy Chain for Gene Therapy of Hemophilia A
`L Chen, II Lu, J Wa11g, R Sarkar, X Ya11g, II Wang, KA lligh and W Xiao
`Biochemical Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency
`Following Adena-associated Virus Gene Therapy
`fl Merritt/I, T Nguyc11, / Do11ic/1, D Matcm 011d DB Schowalter
`
`ACQUIHED AND MULTIGENIC DISEASE
`Acid Ceramidase Upregulation in Prostilte Cancer Cells Confers Resistance
`to Radiation: AC Inhibition, a Potential Radiosensitizer
`MM Mal1<ly, JC C/1cng, / Li, 5 Elojci111y, WO Meacham, LS Turn er, A Bai, CR Gault,
`AS Mcl'l1crw11, N Garcia, Tl/ Becklwm, A Saad, A Biclawska, / Bielawski, YA Harmun, TE Keane,
`Ml Taha, 1/M l/w11111ouc/a, JS Norri1 011d X Liu
`Activation of Aki as a Mechanism for Tumor Immune Evasion
`Kil Noli, /I I Kat1<J, /II Ki111, 51 l'ai, KY Lin, C-F Hung, T-C Wu and TW Kim
`Extracellular Superoxide Dismutase Is a Growth Regulatory Mediator
`of Tissue Injury Recovery
`JI' Laurila, MO C111te//01w, A Curcio, L[ Lcwtikai11c11, M llcwparc111ta-Soli11, T/ Gro11roos,
`I' Mwjamaki, S Martikoi111'11, M 5011/oro 011d MO Loukka11c11
`RNA Interference Targeting STIM 1 Suppresses Vascular Smooth Muscle Cell
`Proliferation and Neointima Formation in the Rat
`re Auliwt, Y 51111i, A Coulo111lw, N Mouyc110I, C Vriy11aud, I' lcpri11ce, I' Lccl,at,
`A-M l 011111r1; 011// J-S /Iulo/
`
`VECTOR ENGINEEHING AND O[1.IVEHY
`High-efficiency Transduction of the Mouse Retina
`l>y Tyro\im•-mut,mt AAV Serotype Vectors
`II l'ct11-Silva, A /Ji11c 11l,•1c 11, () Ii, \-II flli11, V Cl,ioclo, /-/ l'C111<J, I ll1011y, 5 lolot11khi11,
`A .\livmtova, A\ I c'IVi11 1111c/ WW //c1111wi11/1
`l.fficiP11t l11tr,1tliy111ic Gt•1w lr,1mfer rollowi11<J /11 Situ Ad111i11istratio11
`of ,I rAAV Sl'rnlypt' B Vl'ctor in Min• ,111d No11ln1111,111 Pri111,1tes
`A /\t,11,·,111. II \Ii, ,·111,·, I /J11h1,·il, 0 ,\cl1C1li, c; l'otf,,vi11, C /mc/1/1'/, /\' /Jc•1c/HJ1llf)I, /J kl11l111101111,
`\' (!1,·1d, N /11y/111, I' f..111111/i,·1 1111cl \I\ /i111111, ·1111e11111
`
`Th is ma t eria .1 w as cop ied
`3t the NLM a nd ma ybe
`5'ubject USC.op.yr ig;ht Law s
`
`

`

`contents
`
`Molecular
`___ Th era Ry: ______________ ___________
`
`480
`
`491
`
`500
`
`508
`
`516
`
`524
`
`538
`
`548
`
`554
`
`562
`
`570
`
`Combinatorial Evaluation of Cations, pH-sensitive and Hydrophobic Moieties
`for Polymeric Vector Design
`SY Wong, N Saad and D Putnam
`
`Image-guided, Lobe-specific Hydrodynamic Gene Delivery to Swine Liver
`K Kamimura, T Suda, W Xu, G Zhang and D Liu
`
`Selective Enhancement of the Uptake and Bioactivity
`of a TAT-conjugated Peptide Inhibitor of Glycogen Synthase Kinase-3
`AP Manceur, BO Driscoll, W Sun and J Audet
`
`VECTOR TOXICOLOGY, IMMUNOGENICITY AND SAFETY
`Cancer-induced Expansion and Activation of CDl 1 b·Gr-1 • Cells Predispose Mice
`to Adenoviral-triggered Anaphylactoid-type Reactions
`K Pande, R Ueda, T Machemer, M Sathe, V Tsai, E Brin, MJ Delana, N Van liaoijm,
`TK McC/anahan, JE Talmadge, LL Ma/clawer, JI/ Phillipi and OM LaFace
`
`Detection of Intact rAAV Particles ur to 6 Ye,1rs After Successful Gene Tr,msfer
`in the Retina of Dogs and Primates
`K Stieger, J Schraeder, N Provost, A Mendei-Madeim, /J Bcl/Jc/laa, G Le Mc11r, M Wl'lwr,
`/· Y Deschampi, B Lorenz, P Mou/lier and F Ila/ling
`
`Striatal Readministration of rAAV Vectors Reve.ils an Immune Resronse
`Against AAV2 Capsids That Can Be Circumvented
`CS Peden, FP Manfredsson, SK 1/eirmnider, AE Poirier, C Burger, N M111yuku and Ii/ Mandel
`
`OLIGONUCLEOTIDE THERAPEUTICS
`Rational Design Leads to More Potent RNA Interference Ag.iinst Hepatitis B Virus:
`Factors Effecting Silencing Efficiency
`K Keck, EM Volper, RM Spengler, DD Lang, CY Chan, Y Ding and AP McCuffrcy
`
`Guidelines for Antisense Oligonucleotide Design .ind Insight
`Into Splice-modulating Mechanisms
`A Aartsma-Rus, L van Vliet, M 1/irschi, AAM Janson, II lfcc1111kcrk, CL de Winter, S clc Kirnpc·,
`Judith CT van Deutekorn, Peter AC 't ffaen and G-/8 von Orn111en
`
`Design of Phosphorodiamid.ite Morrholino Oligorners (PMOs) for the Induction
`of Exon Skipping of the Human OMO Gene
`LJ Popplewell, C Trallet, G Dickson and Iii Gml,am
`
`CELL THERAPY
`Inhibition of Multidrug-resistant Acinctobactcr haunwnnii liy Nonviral f:xpres\ion
`of hCAP-18 in a Bioengineered Human Skin Tissue
`CL Thamas-Virnig, JM Centanni, CE Johnston, L-K lie, 5/ Sdrlo11c1; Kr Von Winklc, // C/w11,
`AL Gibson, A Szilagyi, L Li, R Shankar and Ell Allcn-l loff111an11
`
`Human Neural Stem Cells C.in Target and Deliver Therapeutic Genes
`to Breast Cancer Br.iin Metastases
`KM Joa, Ir/ Park, JY Shin, J Jin, BG Kang, Ml/ Kim, 5/ Lee, M Jo, SU Kim one/ D-11 Na111
`
`Th is materia l w as.cop,ied
`3tth,e NLM and may b,e
`5'ubject USCop,yright L3ws.
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`original article---- - - -
`
`---···---·-·- .. __________________ ..... ·····-· ...... .
`
`Design of Phosphorodiamidate Morpholino
`Oligomers (PMOs) for the Induction of Exon
`Skipping of the Human DMD Gene
`Linda I Popplewell', Capucine Trollet1, George Dickson 1 and Ian R Graham 1
`
`'School of Biological Sciences, Royal Holloway-University of London, Egham, UK
`
`Duchenne muscular dystrophy (DMD) is caused by out(cid:173)
`of-frame mutations of the human DMD gene. Antisense
`oligonucleotides (AOs) have previously been used to
`skip additional exons that border the deletions such that
`the reading frame is restored and internally truncated,
`but functional, dystrophin expressed. We have designed
`phosphorodiamidate morpholino oligomer (PMO) AOs
`to various exons of the human dystrophin gene. PMOs
`were designed to have their target sites overlapping
`areas of open RNA structure, as defined by hybridiza(cid:173)
`tion-array analysis, and likely exonic splicing enhancer
`(ESE)/silencer sites on the target RNA. The ability of each
`PMO to produce exon skipping was tested in vitro in nor(cid:173)
`mal human skeletal muscle cells. Retrospective analysis
`of design parameters used and PMO variables revealed
`that active PMOs were longer, bound to their targets
`more strongly, had their target sites closer to the accep(cid:173)
`tor splice site of the exon, overlapped areas of open con(cid:173)
`formation (as defined by the hybridization or the RNA
`secondary structure prediction software), and could
`interfere with the binding of certain SR proteins. No
`other parameter appeared to show significant associa(cid:173)
`tion to PMO-skipping efficacy. No design tool is strong
`enough in isolation; however, if used in conjunction with
`other significant parameters it can aid AO design.
`Received 4 July 2008; accepted 25 November 2008; publis/1ed on/inc
`13 January 2009. doi: 10. 1038/ mt.2008.287
`
`INTRODUCTION
`Duchenne muscular dystrophy (DMD) is a severe X-linked
`muscle-wasting disease, affecting I of 3,500 boys. Prognosis is
`poor: loss of mobility by the age of 12, compromised respiratory
`and cardiac function by late teens, and probable death by the age
`of 30. '!he disease is caused by mutations within the large DMD
`gene such that the reading frame is disrupted, leading to lack of
`dystrophin protein expression and breakdown of muscle fiber
`integrity.' 'lhe [)Ml) gene is large, with 79 exons. '!he most com(cid:173)
`mon DMD mutation is genomic deletion of one or more exons,
`generallr centered around hotspots involving exons ,J,t- 55 and the
`5'end of the gene. 2 /vlutations of the /),\1/) gene that preserve the
`
`reading frame result in the milder, non-life-threatening Becker
`muscular dystrophy.
`Exon skipping induced by antisense oligonucleotides (i\Os),
`gencr,1lly based on an IlNA backbone, is a future hope as a ther(cid:173)
`apy for DMD. Indeed, by skipping out-of-frame mutations of the
`/Ji'v/1) gelll:, the reading frame can be restored and a truncated.
`yet functional, Becker nrnsrnlar dptrophr - like dystrophin pro ·
`kin is expressed. Studies in hu111an (dis i11 1·itro'' and in ani (cid:173)
`mal modl'ls of thl' disease i11 1·i1·0· ·• havl' proven thl' prinl"ipk
`of exon skipping as a potential thnap)' for ()/\!() (rl'l'il'\\'l'd in
`ref. 10). Initial clinical trials using two dill'erl'nl AO che111istrks
`I phosphorodia111idate morpholino oligomn ( 1'/\10) (F /\luntoni,
`personal co111111unication) and phosplwrothioate-linkl'd 2'-0 -
`ml'thyl HNA (2'0MePS)J 11 havl' rl't:l'ntly bec.:n performl'd, with
`encouraging results. Indisputably impressive.: rl'sloration of
`dystrophin expression in thl' TA 111uscle of four ()Ml) patients
`injected with a 2'( Hvkl'S AO to exon :i I has hl'c.:n reportc.:d by
`van ()c.:utekom ct 11/. 11
`I lowl'ver,
`it should he noted that, relative to 2'01\kl'S
`AOs, l'MOs have hl'en shown to prnducl' more: u111sistent and
`sustained exon skipping in thl' 111r/x moUSl' modl'I of 1)/\11)
`(rc.:fs. 12- l•l and A. Malerha cl "'·• nia1111script submitll'd}, in
`hun1a11 muscle l'xplants, 1 • and in drtrophic caninl' cells i11 l'itm. '"
`Most important, I'M Os have t·xcclknt safety profiles from clinical
`and predinical data. 1'
`'!he first step to a clinical trial i, the choice of thl' optin1al
`AO target sill' for skipping of thme /),\//) cxons 111ml com(cid:173)
`monly deleted in I >Ml). ln -dl'pth analris of arrars of 2'0~kl'S
`AOs have been reportcd, 1" ·' '' and relatic>11,hips between skipping
`bioactivity and AO variables examined. I lnl', we report the first
`detailed study of the role that A<> target -site variables hal'e 011 the
`hioactivity ofl'MOs to induct: skipping. '!he results reported hnc
`should have an impact on the.: initial planning and design of anti •
`sense l'MOs for future potential clinical trial,.
`
`HESUL TS
`PMO design and analysis of bioactivity
`A unique sc.:t of66 l'MOs has been dc.:signed to target ernn, •l·l. ·1:i ,
`'16, 51, and 53 of the human gene !'or drstrophin . ·1 hl· lk,ign pn >CL'S\
`for exon 53 is depicted in Figure I, and has al,o bcl'll pnl'onncd
`for the other four l'X<lllS (data not shC11rn). '!he cxon ,equL'nCL' \\'a,
`
`Com·spoml,·11r,·: Ion R Gmlw111, School of /Jio/oyirn/ Science's, lloyol 1/ollowoy- University of Lone/on, [y/1u111, Su11cy, IW20 01 X, llK.
`E-mail: i.grohar11(nlr/1ul.ac.uk
`
`55•1
`
`Th is mat e rial w ascopie<I
`3tthe NLM .3n,:I m ay b>e
`S<ubj ect US Copyright. L3WS
`
`

`

`, l /1,· ;\1111•111 .1n
`
`Design of PMOs for Skipping of DMD gene
`
`I a sc3s c sF21AsF □ BACA 1 □ sRp4o □ sRpss I
`
`' I -
`I
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`-6
`
`>,
`i"
`ro
`C
`Q
`"' N
`i I
`e Publi:;hocl 2'-OMo AONs
`
`Exon 53 bases 407-618
`440
`420
`
`100%
`
`460
`
`480
`
`500
`
`520
`
`540
`
`560
`
`580
`
`600
`
`· · · · · · · h53AON1 (+)
`
`---h53AON2 (-)
`
`25 mcrs
`
`30 mors
`
`riqur(' 1 Sc ht•mt· \1m1111.1ri1imJ tht• tools med in the dc\i()n of PMO\ to cxon 53. (a) Results of ESEfinder analysis, showing the location and values
`<1bove threshold for SF2/ASF, SF2/ASF (BRCA 1 ), SC35, SRp40, and SRp55, shown as gray and black bars, as indicated in the legend above. (b) Output
`of PESX ilnalysis, showing the location of exonic splicing enhancers as solid lines, and exonic splicing silencer as a dashed line. (c) Rescue E_SE an_aly(cid:173)
`sis for exon 53, showing predicted ESEs by lines, and where they overlilp, by a ladder of lines. (d) AccessMapper analysis of in vitro hybridization.
`Synthetic pre-mRNA containing exon 5 3 ilnd surrounding intrans subjected to a hybridization screen against a random hexamer oligonucleotide
`arrily, as described in Milteriills Jnd Methods section. Areils of hybridiziltion, suggestive of areas of open conformation, are indicated by pea_ks on the
`graph. (c) The position of the target sites of two 2'OMePS AOs studied previously'" are shown for comparison. (f) The location of the target sites for all
`the 25 mer and 30 mer PM Os to cxon 5 3 used in this study are indicated by lines, and numbered according to the scheme used in Supplementary
`Table Sl, except for exclusion of the prefix "1153."
`
`analyzed for the presence of exonic splicing enhancers (ESEs) and
`exonic splicing suppressors or silencers and the outputs aligned
`for the three available algorithllls, ESEfinder (Figure la),2 11
`21 PESX
`·
`(Figure lh),1 2!' and llcsrne ESE (Figure lc). 21 I lybridization(cid:173)
`array analysis was also perforllled for e,Kh exon i11
`l'ilro, as
`described in Materials and Methods section. 'I he peaks shown in
`Figure Id indicate areas of the exon that are in a conforlllalion
`able to hybridize to the array, and which Ill.I)' consequently prove
`more accessible to antiscnse A( )s. 'I he coincidence of ES Es, as pre(cid:173)
`dicll'd by two or more algorithllls and hybridization peaks deter(cid:173)
`mined expcrilllentally, was used to design arrays of 25 mer and,
`subsequently, 301ller P/\1Os, the positions of which are shown in
`Figure If. 'I he binding sites liir 2'Oi\kl'S A Os described previ(cid:173)
`ously'" are shown ltn comparison (Figmc le).
`Ead1 l'/\H) was tested in primary cultures of human skektal
`nn1scll'. int riplicale, in al least 1,rn expnilllents, and on-r a range of
`rnnrl'lll rat ions from 50 lo '.,()() nmol/1. 'I heir hioadivil y was deter(cid:173)
`mined by rt'\'l'IW trans,ript ion P< :ll .111.dysis, whkh showcd a wide
`variation in the krcl of l'Xon skipping ind11red (Supplcml'lltary
`
`Figure SI, and data not shown), ranging, for example, for the
`targeted skipping of exon 53, from 0% for h53CI (Figure If and
`Supplementary Figure SI, lane 2) to 80% for h53AJ0/3 (flgure If
`and Supplementary Figure SI, lane 6). Often when eflicienl skip(cid:173)
`ping of the targeted exons was achieved, an additional high molec(cid:173)
`ular-weight product that is slightly shorter than the full-length
`product was seen (Supplementary Figure SI, lanes 4 and 5). 'I his
`has been reported previously and is attributable to heteroduplex
`formation. 1·2'' Sequencing of the PCR products verified accurate
`skipping of the targeted exon (data not shown). 'I he activity of
`each l'MO at the stall'd optilllal concentration is sulllmarized in
`Supplementary Tahlc SI, 1\ioactivity is expressed as a percentage
`of the skipped am pl icon relative to total PCll product, as assessed
`by densitometry. Specific, consistent, and sustained exon skipping
`was evident for •1·1 of the 66 l'MOs tested.
`
`In silica analysis of PMOs
`Wt• tht•n perforlllcd a retrospective i11 silico analysis of the charac(cid:173)
`ll'ristics of all (16 l'MOs tested in this study, with respect to PMO
`
`Moll'rn/,11· ·11,..,.,,,,,. 111I. I'/ 11<1 .. 1111.11. .'1111'1
`
`This mate rial was rnpcied
`at the NLM and may b.e
`~ub.ject USCopcyright Laws
`
`

`

`Design of PMOs for Skipping of DMD gene
`
`Figure 2 Mfold secondary structure prediction for 1•xo115 I of th, · hurn,rn f)/1,,71) 11,·11t ·. MfOLD a11,11y\h1'' W,1\ p1 ·rlor1rn,d millCJ PXOll 53ph1s 50111 of tilt•
`upstream and downstream intrans, and with a maximum l.i.isc-p,1iri119 dist,111Cc of I 00111. ·1 hi' i11tro11 ,ind exon bo1111d,1riPs ,He• i11dicat1·d, ,1\ ,1rc th1• pmit iom
`of the target sites of the l.iioactive PMO h53A30/2, and an inactive PMO (1153112). [x,1111ples ul open and timed HN/\ setond,iry stnrctwe .ire• ,irrowc•d.
`
`length, the distance of the !'MO-target site from the splice donor and
`acceptor sites, !'MO-to-target binding energy and l'MO-to-l'MO
`binding energy, as calculated using l{NAstructure2.2 sofiwarc for
`the equivalent RNA- RNA interaction, and percentage GC content
`oft he l'MO, the results of which arc sunHnarizcd in Supplementary
`Table SI. Also shown in Supplcmcnlarr 'fohlc SI is the percent(cid:173)
`age overlap of each !'MO-target site with sequences shown to be
`accessible to binding as determined experimentally by the hexamcr
`hybridization-array analysis. ·1 he relationship hclwccn !'MO-target
`site and !{NA sL·condary structure was also examined using the pro(cid:173)
`gram MFOL()'" (Figure 2 and data not shown}, with the percentage
`overlap of l'MO-largcl silc with sequence predicted lo he in open
`conformation by M FOi.i) analysis givrn in Supplemcnlary 'fohlc
`
`SI. ESEfinder and SSF (hllp://www.11111d.be/SSF/) sotiware anal\'·
`sis of exon sequences revealed the pmition\ of putative SI{ pr;> ·
`lein - hinding motifs (SF2/ASF (hy two algorith1mJ, SC:J5, SHp-10,
`Sl{p55, Tra2ll, and 9G8). 'I he highest score over threshold for each
`SH protein is given for each PMO in the columm on the right of
`Supplementary 'fa hie SI. A !so shown is the degree of o\'crlap of
`each PMO-largel site with the ESE and exonic splicing suppres\or
`or silencer regions predicted by Hesrnc ESE and l'ESX.
`
`Statistical analysis of design parameters
`in relation to PMO l>ioactivity
`For this slalislical analysis, liioacliw l'i\ll h arr (1111,idnrd lo hl'
`those that produce owr :,•:;, skipping. ll'hilc tlio,l' that produ,·l'
`
`556
`
`This m aterial w as copied
`at the NLMand may b-e
`'>u.bj ect USCopyright Laws
`
`lt ' lt 'll', l/l(t/('('ll/11r1/1n111•r.1 •1g "'' · I / ""· \ 111 ,11. .' 1111•1
`
`

`

`Table 1 The correlation of significant design parameters and PMO-target site properties to skipping efficacy
`
`Design of PMOs for Skipping of DMD gene
`
`Distance from
`acceptor site
`
`% overlap with % overlap with strongest % overlap with
`hybridization peak
`hybridization peak
`BRCAl motif
`
`0.056
`
`0.045
`
`0.258
`
`0.003
`
`0.002
`
`0.46
`
`0.026
`
`0.091
`
`0.261
`
`% open
`PMO-target
`binding energy conformation
`
`(l.001
`
`0.094
`
`Length
`
`0.017
`
`0.004
`
`<0.001
`
`0.025
`
`0.002
`
`0.003
`
`-0.618
`
`0.275
`
`0.545
`
`-0.421
`
`Comparison
`
`Ineffective \'ersus
`eflectiw
`
`]netlecti\'e \WSUS
`
`> 75 1}(, skip
`
`Spearman\
`correlation
`codlicient
`
`Spearman\/' value
`
`0
`
`0.0259
`
`0
`
`0
`
`0.0363
`
`0
`
`0.0341
`
`To establish the significance of design parameters and phosphorodiamidate morpholino oligomer (PMO) target-site properties to bioactivity, Mann-Whitney rank
`sum test analysis was performed for each, comparing inactive PMOs to all effective PMOs, and to those that are most effective (i.e., over 75% skipping). Criteria with
`Pvalues <0.05 in one or more comparisons are shown. The correlation of these variables to bioactivity is confirmed by Spearman's rank-order test analysis of the entire
`data set, for which Spearman correlation coefficients and P values are given. Sizes of data groups are: ineffective, n = 22; effective, n = 44; 75-100% skip, n = 10.
`
`<5% skipping arc rn11sidcrcd inactive. For each of the parameters
`listed in Supplementary Table SI, rn111pariso11 was made between
`bioactivc and inactive l'MOs using the 11011paramctric Ma1111-
`Whit11cy rank su111 test, or, when ii was statistically valid to do so,
`the parametric Student's /-test (two-tailed). 'lhc I' values obtained
`fro111 such analyses arc listed in Tahlc I l<ir the para111ctcrs that
`show varying levels of sig11ilica11cc to bioactivity. C:011sidcri11g the
`data as a whole, the variable that showed the highest significance
`to l'MO hioactivity was the hi11di11g energy of the l'MO to the
`cxo11 (/' = (l.00 I); the most bioactivc l'MOs arc predicted to hind
`helter to their targl'l sites. 'I hose I'M Os that overlap with peaks
`identified hy the expcri111ental hybridization-array analysis arc
`not sig11ilica11tly more active than those that do 1101 (/' = 0.056),
`but when only the strongest peak for each cxon is considered,
`this para111ctcr bcco111cs highly significant (/' = 0.003). Distance
`of the !'MO-target site to the splice acceptor site of the cxon was
`also significant (/' = O.O(H), with l'MOs whose target sites were
`closer to the acceptor site being 111orc active. l'MOs whose target
`sites showed coincidence with hi11di11g motil\ for the Sil protein
`SF2/ASF (as defined by the llllC:AI algorithm of Smith ct a/.! 1
`produced sig11ilica11tly greater skipping(/'= 0.026), but only when
`the data is considered as a whole. l'MO length is also a significant
`paran1ctcr (/' = 0.017), with longer l'MOs being more clfrctivc at
`inducing skipping. Box plots of the significant variables identified
`here arc shown in Supplementary Figure S2. None of the other
`variables rnnsidned in this study wne shown to have any sig11ili(cid:173)
`c111cc to A() hioactivity.
`"Ii> ascertain which parametns/desig11 tools arc the most
`powerful, we also used the /\la1111--Whit11cy rank sum test to com(cid:173)
`pare the most active 1'1\IOs (i.e., those that induce >75% skip(cid:173)
`ping of the target exon) to those that were i11adive (i.e., those
`that produce <5% skipping), and the /' values obtained from
`such analyses arc listed in Tahlc I. lloxplols of the sig11ilica11t
`variables 1<11· this comparison arc shown in Figure J. 'lhcrc is
`strong sig11ilica11n· of overlap of the l'i\l() large! site with the
`strongest hybridization peak for l'itlh l'XOll (/' 0.002); more of
`the most hioarli\"e l'i\H ls had their large! sill's lOit1lide11t with
`seq11c11n·s al"l"essihlc Io bi11di11g th;111 thosl' Iha! were inaL"lil'l'.
`·1 his is rl'illl<ll"l"l'd by the obsn\"alio11 th;tl till' target sill's of 1'1\1( )s
`that prnd11l·l·d m·n 75 11
`11 skippi11g overlapped sig11ili,·a11tly 111\lll'
`llN,\ that was i11 opc11 lOlll<llt1talio11, rl'l,1tiYl' to i11arti\"l' 1'1\1( )s
`
`)
`
`(/' = 0.025). Stronger binding between the PMO and its target
`exo11, PMO length, and proximity of the target to the acceptor
`site arc also significant parameters when co111paring the most and
`least effective reagents.
`Spearman's rank-order correlation was used to establish
`potential relationships between design parameters and skip(cid:173)
`ping bioactivity using the entire set of PMOs (sec Table I). 'I his
`shows statistically significant correlations between skipping
`bioactivity and !'MO-target-binding energy (r, = -0.618, P = 0),
`percentage open conformation (r, = 0.275, I' = 0.0259), PMO
`length (r, = 0.5·15, I' = 0), distance fro111 the splice acceptor
`site (r, = -0.421, I'= O), percentage overlap with the strongest
`hybridization peak (r, = 0.46, /' = 0), and overlap with an exonic
`splicing suppressor or silencer sequence, as predicted by PESX
`(r, = 0.261, /' = 0.(1348).
`
`Linear discriminant analysis
`'I his analysis was performed on all possible mm bi nations of
`length, overlap with the SF2/ ASF (BRCA 1) motif, percentage
`overlap with areas of open conforma