Molecular therapy.
`v. 17, no. J (f\1::lr. 2009)
`Gcne:c:11 Coi:Act1or.
`\N1 r.t10·1975r-;,
`
`.
`
`1
`~ ' " ' .lL ~ --:u ar
`
`vol. 17 no. 3 march 2009
`www.1110/cculartl,cmpy.org
`
`5
`
`1 ;
`
`?~
`
`officinl jounwl of the
`American Society of
`Gene Therapy
`
`Bioengineered skin:
`working the bugs out
`Exploiting miRNAs for vector engineering
`
`Neural stem cells target brain metastases
`
`' I
`
`:~~~h::~~.i:,~::ac;~ed ~ ¥~~i&PY
`
`AMERICAN
`SOCIETYof
`
`nature publishing group •
`
`

`

`Molecular
`______ Th era J?-Y-_11_1,_11_a_1.n_,_o_le_c_11_1t_,r_t_l,_e,_·,_1p_y_.<_J1_Jt __________________________________ ~ -
`
`Molecular Therapy is published by Nature Publishing Group, a
`division of Macmillan Publishers Ltd on behalf of the American
`Society of Gene Therapy.
`
`SCOPE
`
`Molecular Therapy is the monthly publication of the American Society of Gene
`Therapy (ASGT). The journal publishes original scientific papers in the areas of
`gene transfer, gene regulation, gene discovery, cell therapy, experimental mod(cid:173)
`els, correction of genetic and acquired diseases, and clinical trials. Manuscripts
`describing new methodological advances will also be considered for publica(cid:173)
`tion. In addition, Molecular Therapy publishes timely reviews, commentaries,
`and scientific correspondence. Although Molecular Therapy is the official journal
`of ASGT, it is international in scope and publication. The major criteria for ac(cid:173)
`ceptance and publication of a manuscript are originality, high quality, scientific
`rigor, and interest to a wide audience of readers.
`
`This journal is covered by AIDS Abstracts, BIOBASE, Biotechnology Citation In(cid:173)
`dex, Chemical Abstracts, Current Contents, Excerpta Medica, Abstract Journals,
`lnpharma Weekly, Index Medicus/MEDLINE, Pharmacogenomics and Outcomes
`News, Reactions Weekly, EMBase, EMBiology, and Scopus.
`
`EDITORIAL
`
`All correspondence should be addressed to: Robert Frederickson PhD, Editor for
`Molecular Therapy, 214 Summit Avenue East, Suite 305, Seattle, WA 98102-
`5640. Tel/Fax: 206-724-7760. Email: editor@molther.org. All manuscripts
`should be submitted online at: https://www.editorialmanager.com/mthe/.
`
`PUBLISHER
`
`All business correspondence and inquiries should be addressed to: Molecular
`Therapy, Nature Publishing Group, 25 First Street, Suite 104, Cambridge, MA
`02141. Tel:+ 1 617 475 9221. Fax:+ 1 617 494 4960.
`
`Publishing Manager: Elizabeth Durzy Senior Production Editor: Anthony Dunlap
`Publishing Assistant: Caitlin Stier
`
`SOCIETY
`For information, contact the American Society of Gene Therapy at:
`Tel:+ 1 414 278 1 341. Fax+ 1 414 276 3349; E-mail: info@asgt.org;
`Web: www.asgt.org.
`
`2009 SUBSCRIPTIONS
`i11stit11tio11al ml1scripti1111s
`NEW INSTITUTIONAL POLICY: Nl'G llds moved lo ,1 1ite licemc policy for
`institutional online access, usirHJ prices b,,1Pd 011 rt1II-Time [quiv,Mnls (FTE)
`or Research and Development (H&D) 11,,rr. l111lilt1l iom m,iy al\O pt1rcha1c a
`separate print subscription.
`
`SUBSCRIBING TO A SITE LICENSE: Co11t,1ct your local s,1lc1 representative for a
`tailored price quote for your i111titt1lion. You will he rcqt1ircd lo complete a Nl'G
`site license agreement. More information, contact details and FTE/H&D defini(cid:173)
`tions are available at the http://natt1re.com/librarics.
`
`INSTITUTIONAL PRINT SUBSCRIPTIONS: Orders can be pl,1ced with your
`regular subscription agent or through NPG-eilher online or by contacting our
`customer service department. Prices are as follows: The Americas $1 ,669.00,
`Europe El,436.00, Japan ¥245,600.00, UK/ Rest of World £927.00.
`PERSONAL SUBSCRIPTIONS: Personal customers who pay by personal check
`or credit card can either purchase a combined print plus online subscription or
`an online only subscription. Prices are as follows: Combined (print plus online)
`The Americas $556.00, Europe (484.00, Japan ¥81 ,900.00, UK/ Rest of World
`£369.00. Personal (online only) The Americas $501.00, Europe €437 .00, Japan
`¥73,700.00, UK/Rest of World £278.00.
`
`CONTACT INFORMATION
`site licenses
`
`THE AMERICAS: Tel: + 1 800 221 2123. Fax: + 1 212 689 9711.
`E-mail: institutions@natureny.com
`
`ASIA PACIFIC (excluding South Asia, Australia and New Zealand): Tel: +81 3
`3267 8769. Fax: +81 3 3267 8746. E-mail: institutions@natureasia.com
`
`AUSTHALIAAND NEW ZEALAND: Tel: +61398251160. Fax: +6139825 1010.
`E-mail: nature@macmillan.com.au
`
`INDIA: Tel: +91 124 288 1054. Fax: +91 124 288 1053.
`E-mail: npgindia@nature.com
`
`THE REST OF THE WORLD: Tel: +44 (0) 20 784 3 4759. Fax: +44 (0) 20 7843
`4998. E-mail: institutions@nature.com
`
`pri11t s11bscriptio11s (i11c/11tli11g si11glc iss11c p11rcl,11ses)
`ALL CUSTOMERS (excluding Japan, Korea and China): Customer Service
`Department, Nature Publishing Group, Houndmills, Basingstoke, Hants, RG21
`6XS, UK. Tel: +44 (0) 1256 329 242. Fax: +44 (0) 1256 812 358.
`E-mail: subscriptions@nature.com
`
`JAPAN, KOREA AND CHINA: Nature Publishing Group, Nature Japan, Chiyoda
`Building 2-37, lchigayatamachi, Shinjuku-ku, Tokyo 162-0843, Japan. Tel: +81 3
`3267 8751 . Fax: +81 3 3267 8746. E-mail: institutions@natureasia.com
`
`Prices are applicable in the following regions: US dollars($) for North, Central,
`South America and Canada; Euros (€)
`for all European countries (excluding the
`UK); Sterling (£) for UK and rest of world; Yen (¥) for Japan. Please ensure you
`use the appropriate currency. All prices, specifications and details are subject
`to change without prior notification. Single issues of M olecular Therapy are
`available.
`
`ADVERTISING: Inquiries concerning print and web advertisements should be
`addressed to: Alf Anderson, Advertising Manager. Tel: + 1 617 475 9231.
`Fax:+ 1 617 494 4960. E-mail: a.anderson@boston.nature.com
`
`SUPPLEMENTS: Inquiries concerning supplements should be addressed to:
`Michelle Libby, Commercial Projects Executive. Tel: + 1 617 475 9230.
`Fax:+ 1 617 494 4960. E-mail: m.libby@boston.nature.com
`REPRINTS AND PERMISSIONS: For reprints of any article in this journal,
`please contact: in North America: Tel: + 1 212 726 9278. Fax:+ 1 212 679 0843.
`E-mail: reprints@natureny.com. Outside North America: Tel:+ 44 (0)20 784 3 4613.
`Fax: + 44 (0)20 7843 4839. E-mail: reprints@nature.com.
`For reproduction rights: ajpermissions@nature.com.
`
`Copyright © 2009 American Society of Gene Therapy, Inc.
`ISSN 1525-0016
`EISSN 1525-0024
`
`All rights of reproduction are reserved in respect of all papers, articles, illustra(cid:173)
`tions, etc. published in this journal in all countries of the world.
`All material published in this journal is protected by copyright, which covers
`exclusive rights to reproduce and distribute the material. No material published
`in this journal may be reproduced or stored on microfilm or in electronic, optical
`or magnetic form without the written authorization of the publisher.
`Authorization to photocopy material for internal or personal use, or internal
`or personal use of specific clients, is granted by Nature Publishing Group to
`libraries and others registered with the Copyright Clearance Center (CCC)
`Transactional Reporting Service, provided the relevant copyright fee is paid di(cid:173)
`rect lo CCC, 222 Rosewood Drive, Danvers, MA 01923, US. Identification code
`for Molecular T/,erapy: 1525-0016/ 07
`
`Apart from any fair dealing for the pt1rposes of research or private study, or
`crilici1111 or review, as permitted tmder the Copyright, Designs and Patent Act
`1988, this publication may he reproduced, stored or transmilled, in any form
`or by any mc<1r1I, only wilh the prior permission in writing of the publisher,
`or in lhc case of rcprographic rcproduclion, in accord,u1n• with tlw trrms of
`licenses isst1C'd by the Copyright Lic!'nsin(J Agency.
`
`Printed on ,Kid-free paper, elfl'Clivc w ith Voh1nll' 15, ls1ue ·1, 2007
`Printed ,md bound in the US by The Sherid,111 l'n•1s, 11,movl'r, l'A, US.
`
`Molernlar rt,erapy (ISSN: 1525-0016) is pt1bli1lwd 11101111,ly liy N,1lure
`Publishing Group, 75 Varick Street, 9th floor, New York, NY 1001 3-191 7.
`Periodicals Posta(Jc paid al New York NY and ,Hldilion,11 m,1ilirHJ olfiC(!I.
`
`Postmaster send address changes to M olcrnlar Tl1Crapy, N,1lure Publishing
`Group, Subscription Dept, 342 llroadway, PM ll 301, New York, NY 1001 3-391 O
`
`While every effort is rnade l>y the publishers to sec that no inacet1ralc or
`misleading data, opinion or statement appears in this journal, they and the
`American Society of Gene Therapy w ish to make it clear that the data and
`opinions appearing in the articles and aclverlisemenls herein arc Ilic respon(cid:173)
`sibility of the contril>utor or adverliser concerned. Accordin(Jly, the American
`Society of Gene Therapy, the publishers and the editors and their respec tive
`employees, officers and agents accept no liability whatsoever for l he consc>(cid:173)
`quences of any such inaccurate or misleading data, opinion or slalenwnt.
`
`

`

`contents
`
`On tire 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.
`
`Molecular
`Therapy vol.17 no. 3 march 2009
`editorial
`The RAC: Double, Double, Toil, and Trouble?
`H Ertl
`
`397
`
`400
`
`401
`
`403
`
`405
`
`409
`
`41 7
`
`425
`
`430
`
`439
`
`448
`
`455
`
`463
`
`4 72
`
`in this issue
`research highlights
`commentaries
`MV9: A Potential Blood-Brain Barrier Buster
`FP Manfredsson, AC Rising and Rf Mandel
`Bioengineered Human Skin: Working the Bugs Out
`L Steinstraesser, S Al-Benno, M Kesling and F Jacobsen
`.
`review
`VECTOR ENGINEERING AND DELIVERY
`MicroRNAs and the Regulation of Vector Tropism
`EJ Kelly and SJ Russell
`
`original articles
`MONOGENIC DISEASE
`Enhanced Factor VIII Heavy Chain for Gene Therapy of Hemophilia A
`L Chen, H Lu, J Wang, R Sarkar, X Yang, H Wang, KA High and W Xiao
`Biochemical Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency
`Following Adeno-associated Virus Gene Therapy
`JL Merritt!/, T Nguyen, J Daniels, D Matern and DB Schowalter
`
`ACQUIRED AND MULTIGENIC DISEASE
`Acid Ceramidase Upregulation in Prostate Cancer Cells Confers Resistance
`to Radiation: AC Inhibition, a Potential Radiosensitizer
`AEM Mahdy, JC Cheng, J Li, S Elojeimy, WD Meacham, LS Turner, A Bai, CR Gault,
`AS McPherson, N Garcia, TH Beckham, A Saad, A Bielawska, J Bielawski, YA Hannun, TE Keane,
`Ml Taha, HM /-lammouda, JS Norris and X Liu
`Activation of Akt as a Mechanism for Tumor Immune Evasion
`KH Noh, TH Kang, JH Kim, SI Pai, KY Lin, C-F Hung, T-C Wu and TW Kim
`Extracellular Superoxide Dismutase Is a Growth Regulatory Mediator
`of Tissue Injury Recovery
`JP Laurila, MD Castel/one, A Curcio, LE Laatikainen, M Haaparanta-Solin, TJ Granroos,
`P Marjamaki, S Martikainen, M Santoro and MO Laukkanen
`RNA Interference Targeting STIMl Suppresses Vascular Smooth Muscle Cell
`Proliferation and Neointima Formation in the Rat
`FC Aubart, Y Sassi, A Coulombe, N Mougenot, C Vrignaud, P Leprince, P Lechat,
`A-M Lompre and J-S Hulot
`
`VECTOR ENGINEERING AND DELIVERY
`High-efficiency Transduction of the Mouse Retina
`by Tyrosine-mutant MV Serotype Vectors
`H Petrs-Silva, A Dinculescu, Q Li, S-H Min, V Chiodo, /·/ Pang, L Zhong, S Zolotukhin,
`A Srivastava, AS Lewin and WW Hauswirth
`Efficient lntrathymic Gene Transfer Following In Situ Administration
`of a rMV Serotype 8 Vector in Mice and Nonhuman Primates
`A Moreau, R Vicente, L Dubreil, 0 Adjali, G Podevin, C Jacquet, JY Deschamps, D Klatzmann,
`Y Chere/, N Taylor, P Mou/lier and VS Zimmermann
`
`

`

`contents
`
`Molecular
`__ _ Thera2y: __________ _
`
`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 Sood 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 Delano, N Van Rooijen,
`TK McC/anahan, JE Talmadge, LL Mo/dower, JH Phillips and OM LaFace
`
`Detection of Intact rAAV Particles up to 6 Years After Successful Gene Transfer
`in the Retina of Dogs and Primates
`K Stieger, J Schroeder, N Provost, A Mendes-Madeira, B Belbellaa, G Le Meur, M Weber,
`J-Y Deschamps, B Lorenz, P Mou/lier and F Rolling
`
`Striatal Readministration of rAAV Vectors Reveals an Immune Response
`Against AAV2 Capsids That Can Be Circumvented
`CS Peden, FP Manfredsson, SK Reimsnider, AE Poirier, C Burger, N Muzyczka and RJ Mandel
`
`OLIGONUCLEOTIDE THERAPEUTICS
`Rational Design Leads to More Potent RNA Interference Against Hepatitis B Virus:
`Factors Effecting Silencing Efficiency
`K Keck, EM Volper, RM Spengler, OD Long, CY Chan, Y Ding and AP McCaffrey
`
`Guidelines for Antisense Oligonucleotide Design and Insight
`Into Splice-modulating Mechanisms
`A Aartsma-Rus, L van Vliet, M 1/irschi, AAM Janson, fl Heemskerk, CL de Winter, S de Kimpe,
`Judith CT van Oeutekorn, l'eter AC 'I lfoen and G-JB van Ommen
`
`Design of PhosrhorocJiamicJate Morpholino Oligomers (PMOs) for the Induction
`of Exon Skipring of the Human DM D Gene
`LJ Popplewell, C Tro/let, G Dickson one/ Ill Groham
`
`CELL THERAPY
`Inhibition of Multidrug-resistant Acinetobacter baumannii by Nonviral Expression
`of hCAP-18 in a BioengineerecJ Human Skin Tissue
`CL Thornas-Virnig, JM Centanni, CE Johnston, L-K lie, SJ Schlosser, Kr Van Winkle, R Chen,
`AL Gibson, A Szilagyi, L Li, R Shankor one/ BL Allen-I loffmonn
`
`Human Neural Stem Cells Can Ta rget ancJ Deliver Therareulic Genes
`to Breast Cancer Brain Metastases
`KM Joo, IH Park, JY Shin, J }in, BG Kong, Mfl Kirn, SJ Lee, M Jo, SU Kirn and 0 -11 Nani
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`original article---------------------------''_J _l l_w_il_m_,,,_,,_,"_' \_"_' ,_,,1_y_ol_l_,,_.,"_' _1,_wr,q,v
`
`Guidelines for Antisense Oligonucleotide Design
`and Insight Into Splice-modulating Mechanisms
`Annemieke Aartsma-Rus1, Laura van Vliet1, Marscha Hirschi1, Anneke AM Janson 2, Hans Heemskerk1,
`Christa L de Winter1, Sjef de Kimpe 2, Judith CT van Deutekom 2, Peter AC 't Hoen 1
`and Gert-Jan B van Ommen 1
`
`'Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; ' Prosensa Therapeutics B. V., Leiden, The Netherlands
`
`Antisense oligonucleotides (AONs) can interfere with
`mRNA processing through RNase H-mediated degra(cid:173)
`dation, translational arrest, or modulation of splicing.
`The antisense approach relies on AONs to efficiently
`bind to target sequences and depends on AON length,
`sequence content, secondary structure, thermodynamic
`properties, and target accessibility. We here performed a
`retrospective analysis of a series of 156 AONs (1 04 effec(cid:173)
`tive, 52 ineffective) previously designed and evaluated
`for splice modulation of the dystrophin transcript. This
`showed that the guanine-cytosine content and the bind(cid:173)
`ing energies of AON-target and AON-AON complexes
`were significantly higher for effective AONs. Effective
`AONs were also located significantly closer to the accep(cid:173)
`tor splice site (SS). All analyzed AONs are exon-internal
`and may act through steric hindrance of Ser-Arg-rich (SR)
`proteins to exonic splicing enhancer (ESE) sites. Indeed,
`effective AONs were significantly enriched for ESEs pre(cid:173)
`dicted by ESE software programs, except for predicted
`binding sites of SR protein Tra2fl, which were signifi(cid:173)
`cantly enriched in ineffective AONs. These findings com(cid:173)
`pile guidelines for development of AONs and provide
`more insight into the mechanism of antisense-mediated
`exon skipping. On the basis of only four parameters, we
`could correctly classify 79% of all AONs as effective or
`ineffective, suggesting these parameters can be used to
`more optimally design splice-modulating AONs.
`
`Received 4 July 2008; accepted 2 7 August 2008; published on/inc
`23 September 2008, doi: I 0, I 038/ml.2008.205
`
`INTRODUCTION
`Antiscnsc oligonuclcotidcs (AONs) arc useful tools to modulate
`gene expression in a sequence-specific manner (reviewed in ref. I),
`Generally, AONs arc used to induce gene knockdown through
`RNase H cleavage of DNA:RNA hybrids of an mRNA . In addi(cid:173)
`tion, mRNA translation can be arrested by stcric hindrance of the
`ribosomal complex by the AON. Finally, AONs can interfere with
`the splicing process to induce nonfunctional mRNAs that arc sub(cid:173)
`jected to the nonsense-mediated RNA decay pathway. Using the
`latter approach, it is also feasible to modulate alternative splicing,
`
`·1 hcsc
`or to block aberrant, disease-causing splice sites (SSs). 2
`mechanisms can be used for studies on developmental processes
`by allowing knockdown of gen es at specific time points,-' or for
`therapeutic purposes. In fact, an RNase H-inducing AON is regis(cid:173)
`tered und er the name Vitravenc to treat cytomegaloviral-induccd
`retinitis, and many AONs aiming at targeted gene downregula(cid:173)
`tion arc in late stage clinical trials mainly as putative anticancer
`drugs. 1 Splice-modulating AONs arc in early phase clinical trials
`for Duchenne muscular dystrophy (DM D). 1 Herc, the modulation
`of splicing (in this case the skipping of an cxon) aims to restore the
`disrupted dystrophin-rcading frame, allowing the generation of
`partly functional proteins and slowing down the severe, progres(cid:173)
`sive muscle wasting phenotype.
`Each antiscnsc mechanism requires stable and cflicicnt bind(cid:173)
`ing of the AON to its target sequence. One obvious determinant
`of AO N efficacy is the accessibility of the target (Supplementary
`Hgurc S l ). Several software programs arc available to predict
`the secondary structure of RNA , of which the 111-fold server
`is the most widely used. ' 'fhis server also provides a so-called
`SS-count for the target sequence, indicating the propensity of a
`nucleotide to be single stranded in a number of potential sec(cid:173)
`ondary structure predictions. ·1 his approach probably reflects
`the actual i11 vivo situation more closely than focusing only 011
`the most energetically stable structure. In addition, the stability
`and binding energy of the AON lo the target sequence inllurnce
`AON cllicienc y. ·1 hi.s depends 011 c,g., AON length and sequence
`constitution and the free energy of local structurcs. 1 To cllicicntly
`hind a largcl scquc1icc, the free energy of the AON-target com(cid:173)
`plex must be higher than that of the largcl complex and that of
`the AON . As AONs arc generally only 17- 2:,-nucleotides long,
`tlwy arc unlikely to fc>rm stable secondary structures. I lowcver,
`most /\ONs can form AON-AON complexes with olhn /\ONs
`of the same sequence (S11pplc111c11lary Figure S2) . ·1 he soft(cid:173)
`ware program llNAstructurc •I.:, has a tool that provides the free
`energy of AON-AON complexes and AON-target complexes, in
`addition to the free energy of individual /\ONs and the largd
`sequence." 'Ihc aforementioned software programs (as well as
`others) can be used lo l'acililalc AON design (reviewed in rcC I).
`Nonetheless, none of them is I 00'¾, conclusive or predictive and
`in general a trial and error procedure is still involved lo identify
`potent AONs.
`
`Correspo111/e11ce: Annemieke Aartsma-Rus, Department of Human Genetics, Leiden University Mcc/ical Center, PO Box 9600, l'<Htzonc 54-1! 2 300 /IC,
`Leiden, The Netherlands . E-mail: a.m.rus@lumc.nl
`
`548
`
`Th is m ate.rial w asco;pied
`at the NLM a nd ma y be
`5'ubj ect USCapyf'ight Laws
`
`1viv1v.11wla11ll1rll1c1·llpy.1Jri: v11 I. 17 1111 . .I, '.i -lH-- '.i '.i.\ 111a r. 2011'1
`
`

`

`Guidelines for Splice-modulating Oligo Design
`
`In addition to more general AON requirements, the differ(cid:173)
`ent applications may introduce further requirements as well. For
`instance, translation-blocking AONs often target the ATG initia(cid:173)
`tion site, whereas splice-modulating AONs target sites involved
`in cxon recognition and inclusion. '] he latter consist of the donor
`and acceptor SSs and branch point sequence, but also include
`exon-internal sequences [exonic splicing enhancers (ESEs) sites]
`that facilitate splicing by binding of so-called Ser-Arg-rich (SR)
`proteins, which in their turn recruit Ul snRNP and U2AF to the
`donor and acceptor SSs, respectivcly. 7 Both AONs targeting (aber(cid:173)
`rant) SSs and ESEs have been shown to be efficient modulators of
`splicing.",., An advantage of exon-internal over SS AONs may be
`that they do not target consensus sequences as ESEs arc weakly
`defined motifs, and that there is a wider range of target choice.
`Numerous software programs predict ESEs, such as ESEfinder
`that predicts binding sites for the four most abundant SR proteins
`(SF2/AS!\ SC35, SRp40, and SRp55), 10
`•11 RESCUE-ESE, 12 and the
`PESX servcr 13 that provide hcxamcrs and octamers, respectively,
`significantly enriched in cxons over introns. "Jhesc programs arc
`implemented in the human splicing finder (http://www.umd.be/
`HSF/), which also provides algorithms to predict binding sites
`for two other SR proteins (9G8 and Tra2[3). In addition, the HSF
`program includes hexamers, octamers, and dccamers associated
`with exonic splicing silencers (ESSs).u 1
`'Ihcse splicing silencers
`are the counterparts of ESEs and prevent exon inclusion. As exon(cid:173)
`intcrnal AONs are hypothesized to work through steric hindrance
`of SR proteins, it is expected that AONs targeting predicted ESEs
`an: more likely to be effective. We have previously analyzed a series
`of I 1'1 exon-intcrnal AONs targeting dystrophin exons using
`ESEfinder and RESCUE-ESE in retrospect, and indeed found that
`effective AONs were significantly enriched in SR-binding sites and
`RESCUE-ESE hexamers."· 1
`' ' We have now extended our series of
`cxon-intcrnal dystrophin AONs to 156, of which 104 (67'¼,) are
`effective and 52 (33%) arc ineffective. In this study, we have ana(cid:173)
`lyzed our larger set for previously analyzed and new parameters to
`
`'
`
`improve AON design guidelines and gain insight into the mecha(cid:173)
`nism of AON-mediated exon skipping.
`
`RESULTS
`AON constitution and thermodynamic properties
`We here compared a series of 156 exon-internal AONs targeting
`exons of the DMD gene transcript. AONs were assigned to either
`the effective (11 = 104) or ineffective (11 = 52) group, depending on
`their ability to reproducibly induce targeted cxon skipping at levels
`of at least 5% as assessed by RT-PCR analysis. Different parame(cid:173)
`ters such as predicted T , length, AON constitution, and thermo(cid:173)
`dynamic properties wcr~ determined for each AON (sec Materials
`and Methods and Supplementary Table SI). 'TI1c values of each
`parameter were compared between the effective and ineffective
`AONs (Supplementary Table S2), and a Wilcoxon signed-rank
`sum test was used to detect significant differences. 13oxplots of the
`parameters that differed significantly between the two groups are
`shown in Figure I.
`First, we compared AON length in both groups. In our series
`AON length varies from I 5 to 25 nucleotides and did not signifi(cid:173)
`cantly differ between the two groups. Second, we evaluated AON
`constitution, as C and G content has been shown to correlate with
`AON-mRNA duplex stability. 17 Indeed, the total number of Gs
`and Cs was significantly higher in effective AONs (P < 0.05). In
`contrast, no significant difference was observed for the percent(cid:173)
`age and total number of A, C, G and U, GA, and GT nucleotides.
`Focusing on the predicted basic melting temperature o:J and '(11s
`using the nearest neighbor model, 1:11
`s were significantly higher
`for the effective group using both calculation methods.
`As a third parameter, we used them-fold program to predict the
`secondary structure of the local target sequence and the AONs, and
`the RNAstructure software version 4.5 to predict thermodynamic
`properties of AONs and AON-target binding. It is expected that
`the ability of AONs to form stable secondary structures with them(cid:173)
`selves or other AONs of the same sequence (see Supplementary
`
`Basic re: ("C)
`
`Nearest neighbours re: ("C)
`
`14
`
`12
`
`10
`
`8
`
`6
`
`2
`
`0
`
`-5
`
`-10
`
`-15
`
`Number of Gcs·
`
`---,-
`
`I
`
`I
`
`-,-
`
`g
`
`I
`
`---'---
`
`---'---
`
`60
`
`50
`
`40
`
`30
`
`'
`
`-+--
`
`---,--
`
`'
`--'---
`
`Effective
`
`Ineffective
`
`Effective
`
`Ineffective
`
`Energy AON-AON complex·
`(kcal/mol)
`
`Binding energy AON-AON ..
`(kcal/mol)
`
`' a 2
`
`----'---
`
`----'---
`
`15
`
`10
`
`5
`
`0
`
`'
`---,--
`
`a g
`
`Etfect1ve
`
`lneffcct1ve
`
`Effective
`
`'
`
`_,_
`Ineffective
`
`70
`
`60
`
`50
`
`40
`
`30
`
`40
`
`-,-
`'
`
`30 g
`
`20
`
`'
`----'---
`
`10
`
`0
`
`---,--
`'
`
`'
`----'-
`
`' I g
`
`---,--
`
`'
`'
`----'-
`
`Effective
`
`Ineffective
`
`Binding energy AON-target'"
`(kcal/mol)
`
`0.8
`
`Local accessibility•
`
`---,--
`
`:
`- -
`
`0.7
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`' B
`' B
`
`'
`'
`'
`
`'
`----'---
`
`'
`' '
`
`'
`' ' '
`'
`----'-
`Effective
`
`Effective
`
`Ineffective
`
`0.1
`
`Ineffective
`
`I iqur,· 1 Boxplol\ of tlw1111<Hly11,1111ic ,rnth,·m<· oli9011ucleotide (AON) parameters that differed significantly between the effective and inef(cid:173)
`f,,,tiv,· qroup. *P < 0.05, **P < 0.005. GC, guanine-cytosine.
`
`,\/olcrn/ar '//1cmpy rnl. 17 no . .I mar. 2009
`
`Th is mat eri a l was co pcied
`at the N LM and may be
`~ubject USCo pcyright Laws
`
`549
`
`

`

`Guidelines for Splice-modulating Oligo Design
`
`"J I lw /\nH'ric dil )o<.i 1 ·I>' ol C< ' lll' ·1 her,1py
`
`Figure S2) will negatively influence their efficacy. As AONs arc rel(cid:173)
`atively short, predicted secondary structures are often not very sta(cid:173)
`ble (6G - 0 kcal/mo!) . No significant difference was observed for
`the free energy of individual AONs. Unexpectedly, for AON-AON
`complexes we observed that complexes formed by effective AONs
`had significantly lower free energy values (and where thus more
`stable) than those of ineffective AONs (P = 0.03). We also used the
`RNAstructure software to predict the free energy of the local target
`structure and the binding energy of AON-target complexes. We
`anticipated that it would be more diflicult to find efficient AONs
`for exons with a stable secondary structure, as these are harder to
`disrupt. 'The free energies of the target exons seem slightly higher
`(less stable) for effective AONs, but this diflerence was not statisti(cid:173)
`cally significant (P = 0.11 ). No difference was observed between the
`free energy of the AON-target complexes. In contrast the binding
`energy of AON-target complexes (free energy of AON-target com(cid:173)
`pared to free energy of target) was significantly higher for effective
`AONs (P = 0.003). This indicates that for splice-modulating AONs
`the efficacy of an AON depends not on the stability of the AON(cid:173)
`target complex, but rather on the amount of energy that is released
`upon AON binding.
`Finally, using SS-count (m-fold server) we determined the
`local accessibility of the target pre-mRNA. We defined the local
`accessibility as the average propensity of target nucleotides to be
`
`single stranded in the predicted secondary structures. We antici(cid:173)
`pated that effective AONs would be enriched in single-stranded
`target sequences, as we hypothesized SR protein binding lo be
`more likely to predicted open secondary structures. Nevertheless,
`we observed that the accessibility of ineffective AONs was signifi(cid:173)
`cantly higher (P = 0.021 ), although both groups targeted sequences
`that were at least partly accessible.
`
`Splicing enhancer and silencer motifs in AON-target
`sequences
`Exon-internal splice-modulating AONs arc hypothesized to act
`through blocking of SR protein binding to ESE motifs. Thus, etlec(cid:173)
`tive AONs are expected to be enriched in ESEs, while the targeting
`of splicing silencer motifs is expected to be counterproductive. In
`addition, we compared the location of the AON-target sequence
`to the SSs, as ESE motifs have been reported to be more active
`when located within 70 nucleotides from either splice side. 18 An
`overview of parameters that differed significantly between effcc-(cid:173)
`tive and ineffective AONs is given in Figure 2, while all results are
`summarized in Supplementary T.tble S2B. Similar to our previ(cid:173)
`ous analysis of a smaller set of AONs, 8 we observed that effective
`AONs arc located significantly closer to the acceptor but not to the
`donor SS. In our current, larger set, the difference became more
`significant (P = 0.006 versus 0.008). For the donor site there was
`
`50
`
`00
`
`50
`
`Nucleotide from 3'-SS'
`---.-
`
`---r-:
`'
`
`20
`
`15 -
`
`10 ·
`
`' !g B,
`
`0 ~ - - - ' - -_____ --'--__ ~
`lrwffcctivo
`Effcclivo
`
`0
`
`SC35 values·
`(with threshold)
`
`SC35 values•
`(relative to threshold)
`
`20
`
`10
`
`0
`
`- 10
`
`- 20
`
`' ' ' ' ' ' g
`
`'
`' '
`' _ l_
`Effective
`
`' ' I
`
`'
`'
`__.__
`
`Ineffective
`
`'
`'
`'
`
`0==----====__,__===-----1
`
`EHuclivo
`
`lnoffoctivo
`
`SRp55 valu es·
`(relative to threshold)
`-,-
`'
`'
`
`' '
`'
`
`' ' g
`
`' '
`'
`'
`'
`--'--
`Effective
`
`g
`
`'
`'
`'
`--'--
`
`Ineffective
`
`PESE values·
`(with threshold)
`
`10
`
`5
`
`0
`
`- 5
`
`-10
`
`-15
`
`-20
`
`-25
`
`80
`
`60
`
`Highost ESEfinclor valuo·
`(with th ros liold)
`~ -- -
`
`Nurn!Jm ol ESEfi11cJer sites'
`(will, thro:;1,olrl) - - -~
`
`RESCUE ESE'
`(number of hoxamers)
`
`14
`
`12
`
`1()
`
`ll
`
`25
`
`20
`
`15
`
`'
`
`'
`
`Ineffective
`
`EJ □ Effective
`
`' '
`'
`' '
`'
`
`'
`
`-,--
`'
`'
`'
`'
`
`l_-'-_~--l
`
`EfhJ C!iV(l
`
`lrH illoclivo
`
`40 Fl ' '
`
`20 ·
`
`to
`
`--r-
`'
`'
`'
`
`---r-
`'
`
`' B
`' B
`
`_ l_
`
`[ffoctivo
`
`lnuffoctivo
`
`Number of Trn 2JI si tus·
`(witt, thro shold)
`
`---,-
`'
`'
`
`-,-
`' '
`
`' B
`
`Effective
`
`lnoffcc tivo
`
`0 -
`
`2
`
`0
`
`14
`
`10
`
`8
`
`6
`
`0
`
`-----,(cid:173)
`
`10 -
`
`' ' ' :BO
`
`Effective
`
`Ineffective
`
`:J
`
`2
`
`0
`
`Number of PESE sites•
`
`10
`
`8
`
`---,-
`
`0 L'::====-------====::__J
`
`Ineffective
`
`Effective
`
`20
`
`15
`
`10
`
`0
`
`Tra2Jl values·
`(with thre shold)
`-,-
`' '
`' ' ' ' '
`
`' '
`
`Ineffective
`
`□ Effective
`
`Figure 2 Boxplots of exonic splicing enhancer (ESE) parmneters of antisense oli<Jo1111clcotidc (J\ON)-t,ir<Jt'l \('<JIH'IIC('\ that dill,·n•d \iq11ili
`cantly between the effective and ineffective group. *P < 0.05. SS, splice site; PESE, putative ESE.
`
`550
`
`Th is mate,ria I w as copied
`at the NLM and may :b,e
`Subject US Copyright: Laws
`
`lVll'll'.1110/a11/11rtl1<'rt1/')'•"'·i: vol. 17 110 .. 1 mar. 200')
`
`

`

`Guidelines for Splice-modulating Oligo Design
`
`a trend toward eflcctive AONs being localed further away to the
`donor site (and thus closer to the acceptor SS, P = 0.09).
`A variety of software programs predicting ESE and ESS motifs
`is available and most of these are implemented in the human
`splicing finder software (http:/ /www.umd.be/HSF/). We analyzed
`th e target sequence of each AON for the presence of ESE and
`ESS motifs and compared the etfoctive and ineffective group as
`described above. No significant difference was observed when
`we analyzed the presence of any predicted ESE in the two groups.
`However, for the vast majority of AONs at least one ESE motif
`was predicted, which might explain the lack of significance. We
`then proceeded to compare individual ESE predicting programs
`starting with ESEfinder and RESCUE-ESE, which previously had
`shown significantly higher values for SC35 and the highest pre(cid:173)
`dicted value for any SR protein (ESEfinder) 8 and significantly more
`hexamers (RESCUE-ESE) 16 in our smaller series of AON. We now
`confirm these findings and for SC35 and the number ofRESCUE(cid:173)
`ESE sites the difference became more significant (SC35: 0.0 I versus
`0.05, RESCUE-ESE: 0.03 versus 0.04), while it became slightly less
`significant for the highest SR value (0.045 versus 0.037). Notably,
`we did not observe the previously identified trend for Sf2/ ASr val(cid:173)
`ues to be higher in the effective group, which bordered on statisti(cid:173)
`cal significance (P = 0.053)." 'Ihis may have been caused by the fact
`that in the previous analysis predicted values without a threshold
`were compared, while here a threshold was applied. ·n1erefore, we
`also determined the values relative to the threshold (either over
`or below) for each of the SR proteins. 'This still did not result in a
`significant increase for SF2/ ASF values in the effective group, and
`the diflcrence became less s