Jl.... VI
`
`offici//1 io11m11/ o( tltc
`;\111crfw11 Sori1;t)' of
`Gc11c T/1cu1p;·
`
`II I
`
`vol. 15 no. 7 july 2007
`11•11•11•. 11wlcc11/a rt licrapy. org
`
`Transport properties of HIV Tat
`Status of cardiov;1suilar gene thn;1py
`,\'/1·1·/>i11g lk,1111;· gets St'vlal{T
`
`PROPERlY or THE
`NATIONAL
`LIBRARY OF
`MEDICINE
`
`Th is m ate.ria I was co p,ied
`at the NUM a nd ma y b,e
`:.u:bject US Qlpyright Law s
`
`naturl' puhlishing group @
`
`

`

`Molecular
`______ Th era )?-y:_w_1_v1_v._11_H_1l_cc_,_,,_11r_t_lt_cr_11c;_p.:...y_.o_rJ:'-'-------------------------------~~-
`
`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 (ASGD. 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
`
`AUSTRALIA AND NEW ZEALAND: Tel: +61 3 9825 1160. Fax: +61 3 9825 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 7843 4759. Fax: +44 (0) 20 7843
`4998. E-mail: institutions@nature.com
`
`print s11bscriptio11s (i11c/11di11g single ismc p11rclt11scs)
`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 (S) for North, Central,
`South America and Canada; Euros (E) 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 im1e1 of Molrwlar Therapy are
`available.
`
`ADVERTISING: Inquiries concerning print and Wl'b advertisements should be
`addressed to: D,1vid B,1gshaw, Advertisement M,rn,1ger, N,1ture Publishing
`Group, 75 Varick Street, 9th Floor, New York, NY 1001 3-1917. Tel: + 1 212 726
`9215. F,1x: + 1 212-696-9591. E-m,1il: d.b,1gshawC<•)natureny.com
`
`SUPPLEMENTS: Inquiries should be ,1ddressl'd lo: T,rn1ara Nicholsor1-tl,H],
`Supplements Editor, Tel: +44 (0) 20 7843 4856. F,1x: +44 (0) 20 7843 4839.
`E-mail: t.nicholson-haqra)n,1ture.com.
`
`All business correspondence and inquiries should be addressed to: Molewlar
`Therapy, Nature Publishing Group, 25 First Street, Suite 104, Cambridge, MA
`02141. Tel: +1617475 9221. Fax: +1617494 4960.
`
`REPRINTS AND P[HMISSIONS: For reprints of ,1ny ,uticlt• in this journ,11, ple,1se
`contact Sharda Tulsic, Tel: +1 212 726 9631. rax: +1212679 0843. E-m.iil:
`s.tulsie@natureny.com. For reproduction ri<]hts: ,1jpl'rrnissiomv•ln,1turc.com
`
`Executive Editor: Elizabeth Durzy Production Coordinator: Tony Dunlap
`
`SOCIETY
`For information, contact the American Society of Gene Therapy at:
`Tel:+ 1 414 278 1341. Fax+ 1 414 276 3349; E-mail: info@asgt.org;
`Web: www.asgt.org.
`
`2007 SUBSCRIPTIONS
`
`i11stitutio1111/ s11bscriptio11s
`NEW INSTITUTIONAL POLICY: NPG has moved to a site license policy for
`institutional online access, using prices based on Full-Time Equivalents (FTE)
`or Research and Development (R&D) staff. Institutions may also purchase ,1
`separate print subscription.
`
`SUBSCRIBING TO A SITE LICENSE: Contact your local sales representative for a
`tailored price quote for your institution. You will be required to complete a NPG
`site license agreement. More information, contact details and rTE/R&D defini(cid:173)
`tions are available at the http://npg.nature.com/libraries.
`
`INSTITUTIONAL PRINT SUBSCRIPTIONS: Orders can be placed with your
`regular subscription agent or through NPG-either online or by cont,1cting our
`customer service department. Prices are as follows: The Americ,11 S 1500.00,
`Europe €1,291.00, Japan ¥194, 100.00, UK/Rest of World £83 3.00.
`
`Copyright iD 2007 American Society of Gene Ther,1py, Inc.
`ISSN 1525-0016
`EISSN 1525-0024
`
`All rights of reproduction arc reserved in respect of all p,1pers, articles, illustr,1-
`tions, etc. published in this journ,11 in all countries of the world.
`
`All m,1terial published in this journ,11 is protected by copyright, which cov(cid:173)
`ers exclusive rights to reproduce ,1nd distribute the rn,1tcri,1I. No m,1terial
`published in this joum.il m,1y be reproduced or stored on microfilm or in
`electronic, optic,11 or m,1gnctic form without the written authoriz,1tion of the
`publisher.
`
`Authorization to photocopy m,1l<'rial for inlern,11 or p<'rson,11 use, or internal
`or pcrson,11 me of specific clients, is qr,rnted by N,1turc Publishing Group to
`libraries ,rnd others re<Jislered with the Copyri<Jht Cltwancc Center (CCC)
`Tr,1111,iction,11 Heporlill<J Servi<<', provided th<' relev,rnt copyright fee is paid di(cid:173)
`rect lo CCC, 222 Holl'wood Drive, D,mvers, MA 01923, US. ldentific,1tion code
`for Molcwlar T/icraJly: 1525-0016/07
`
`Ap.irt from ,1ny f,1ir d,•,1lir1<J for the purposc·s of rc·s!'.ird1 or private study, or
`criticism or n•view, ,11 perr11illl'd und!'r the• Copyri<Jhl, D<'sig111 ,111d Patc·nt Act
`1988, this public,11io11 m,1y be r!'prod11ced, slorc·d or tra111mitlrd, in ,my form
`or by ,1ny llll',lllS, only with th<' prior permis1io11 in writirHJ of the publisher,
`or in tlw c,11e of reprocJr,1phic reprod11ction, in accordance with the t<"rms of
`lin'llll'I issued by the Copyright lice111ing A<J<·rKy.
`
`Print,,d on acid-free paper, effective with Volume 15, ls1ue 1, 2007
`
`Printl'd ,rnd bound in the US by The Sheridan Press, Hanover, PA, US.
`
`PERSONAL SUBSCRIPTIONS: Personal customers who pay by person,11 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 S525.00, Europe €453.00, Japan ¥6B,00.00, UK/Rest of World
`£292.00. Personal (online only) The Americas S472.50, Europe €407.70, J,1p,1n
`¥61,200.00, UK/Rest of World £262.80.
`
`Molcwlar Tlrrrapy (ISSN: 1525-0016) is published monthly by Nature Publish(cid:173)
`ing Group, 75 Varick Street, 9th floor, New York, NY 10013-1917. Application
`to Mail at Periodicals Postage Rates is pending at New York, NY and addition,11
`m,1iling offices.
`Postmaster send address changes to Molecular Therapy, Nature Publishing
`Group, Subscription Dept, 342 Broadway, PMB 301, New York, NY 10013-391 0
`
`CONTACT INFORMATION
`
`site licc11scs
`
`THE AMERICAS: Tel: +1800221 2123. Fax: +1212689 9711.
`E-mail: institutions@natureny.com
`
`ASIA PACIFIC (excluding South Asi,1, Australi,1.ind New 7e,1la11d): Tel: 181
`3267 8769. Fax: +81 3 3267 8746. E-m,1il: i111titutio11s~v11atureasi,1.corn
`
`While every effort is made by the publishers to see that no inaccurate or
`misleading data, opinion or statement appears in this journal, they and the
`American Society of Gene Therapy wish to make it clear that the data ,md
`opinions appearing in the articles and advertisements herein arc the respon(cid:173)
`sibility of the contributor or advertiser concerned. Accordingly, the Americ,111
`Society of Gene Therapy, the publishers and the editors ,md their respective
`employees, officers and agents accept rw li,1bility wh,1t1orver for the consc(cid:173)
`quc11ce1 of any 1uc h inaccurate or misleading d,11,1, opinion or 1t,1te111e11t.
`
`Th is mate ria I was co pied
`atth,e NLM a nd may b-e
`~ubject US Co pyright Laws
`
`

`

`Molecular
`Therapy vol. 15 no. 7 july 2007 - - - - - - - - - - - - - - - - - - -
`
`contents
`
`011 the cover:
`In vivo study of HIV-1 Tat arginine(cid:173)
`rich motif unveils its transport
`properties. See the article by
`Cardarelli ct al. on pages 1313-
`1322.
`
`editorials
`
`1223 NIH Decides Against Continuing NGVLs in Their Current Form
`DA Williams
`
`1223 Remodeling the NGVL Program to Meet Contemporary Needs
`AR Hayward
`
`1226
`
`in this issue
`1227 research highlights
`n1eeting report
`
`1228
`
`Integrating Ideas on lnsertional Mutagenesis by Gene Transfer Vectors
`RM Frederickson
`
`reviews
`
`ACQUIRED AND MULTIGENIC DISEASE
`1233 Current Status of Cardiovascular Gene Therapy
`TT Rissanen one/ S Ylii-1/erttuala
`
`CELL THERAPY
`1248 The Suicide Gene Therapy Challenge: How to Improve a Successful Gene Therapy
`Approach
`C Bonini, A Bondanza, S Kimi Perna, S Kaneko, C Travcrsari, F Ciccri and C Bordignon
`
`original articles
`
`MONOGENIC DISEASE
`1253 Efficacy of Helper-dependent Adenovirus Vector-mediated Gene Therapy in
`Murine Glycogen Storage Disease Type la
`DD Kocbcrl, B Sun, A Bird, YT Chen, K Oka and L Chon
`
`ACQUIRED AND MULTIGENIC DISEASE
`1259 Role of Acid Ceramidase in Resistance to Fasl: Therapeutic Approaches Based on
`Acid Ceramidase Inhibitors and FasL Gene Therapy
`S Elojeimy, X Li11, JC Mckillop, AM EI-Zawa/1ry, D/--1 Holman, JY Cheng, WO Meacham,
`AEM Mohdy, AF Semel, LS T11rncr, J Cheng, TA Day, J-Y Dong, A Biclawska, YA Hann11n and
`JS Norris
`
`1264
`
`Lentiviral Gene Delivery of vMIP-11 to Transplanted Endothelial Cells and
`Endothelial Progenitors Is Proangiogenic In Vivo
`S C/1crq11i, KM Kingdon, C Thorpe, SM K11rian one/ DR Solomon
`
`VECTOR ENGINEERING AND DELIVERY
`1273 Correction of DNA Protein Kinase Deficiency by Spliceosome-mediated RNA
`Tram-splicing and Sleeping Beauty Transposon Delivery
`II Zoycd, L Xia, A Ycric/1, SR Yant, MA Kay, M Puttaraj11, GJ McGarrity, DL Wiest,
`f/S Mcivor, J Tolar and Bil /l/a1ar
`1280 Messenger RNA as a Source of Transposase for Sleeping Beauty Transposon(cid:173)
`rnediated Correction of Hereditary Tyrosinernia Type I
`A Wither, KJ Wangcmtccn, \' C/1en, L Zlwo, ft Fmndscn, JB Bell, ZJ Chen, SC [kkcr, f/S Mcivor
`one/ X Wang
`
`1288 Antisense Oligonucleotide-induced Exon Skipping Across the Human Dystrophin
`Gene Transcript
`S[) Wilton, AM Fall, l'I /lwdiny, G flfcC/01cy, C Coleman and S rtctd1e1
`
`1297 Cellular Dynamics of [Gr lkccplor-T,irgetcd Synthetic Viruses
`K c/c• /l111i11, N llutl,wclt, K 10n C,·11do1/I, ll /lm11in<fc1; I Wm;nc1; M Oqri1 crnd C llrciuc/1/e
`
`Th is m a t e ri a I was « Ipied
`at t he NLM and m a y be
`5ubje,:t US Copyright Law s
`
`

`

`contents
`
`Molecular
`___ Thera2y: _______________ _
`
`1313
`
`1306 Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(/3-amino
`ester)s
`GT Zugates, W Peng, A Zumbuehl, S Jhunjhunwala, Y-H Huang, R Langer, /A Sawicki and
`DG Anderson
`In Vivo Study of HIV-1 Tat Arginine-rich Motif Unveils Its Transport Properties
`F Cardarelli, M Serresi, R Bizzarri, M Giacca and F Beltram
`1323 A Dual Role of EGFR Protein Tyrosine Kinase Signaling in Ubiquitination of AAV2
`Capsids and Viral Second-strand DNA Synthesis
`L Zhong, W Zhao, J Wu, B Li, S Zolotukhin, L Govindasamy, M Agbandje-McKenna and
`A Srivastava
`Lentiviral Vectors Mediate Stable and Efficient Gene Delivery into Primary Murine
`Natural Killer Cells
`J Tran and SKP Kung
`1340 Development and Validation of a Robust and Versatile One-plasmid Regulated
`Gene Expression System
`P Szymanski, Pf Kretschmer, M Bauzon, F fin, HS Qian, GM Rubanyi, RN 1/arkins one/
`TW Hermiston
`
`1331
`
`VECTOR TOXICOLOGY, IMMUNOGENICITY AND SAFETY
`1348 Histone Modifications are Associated with the Persistence or Silencing of Vector(cid:173)
`mediated Transgene Expression In Vivo
`E Riu, Z-Y Chen, H Xu, C-Y He and MA Kay
`
`1356 Comparison of HIV-derived Lentiviral and MLV-based Gammaretroviral Vector
`Integration Sites in Primate Repopulating Cells
`BC Beard, D Dickerson, K Beebe, C Gooch, J Fletcher, T Okbinoglu, DG Miller, MA /aco/J1,
`R Kaul, H-P Kiem and GD Trobridge
`
`1366 A High Throughput In Vivo Model for Testing Delivery and Antivir;il Ellccts of
`siRNAs in Vertebrates
`BD Schyth, N Lorenzen and FS Pedersen
`
`CELL THERAPY
`1373 Bystander Killing of Malignant Glioma by Bone M;irrow-dcrived Tumor(cid:173)
`Infiltrating Progenitor Cells Expressing a Suicide Gene
`H Miletic, Y Fischer, S Litwak, T Giroglou, Y Waerzeggers, I\ Winkelcr, II Li, U 1/immclrcicl,,
`C Lange, W Stenzel, M Deckert, H Neumann, AH Jacobs oficl D von Locr
`1382 Mesenchymal Stem Cells Over-expressing Hep;itocytc Growth Factor Improve
`Small-for-size Liver Grafts Regeneration
`Y Yu, A-H Yao, N Chen, L-Y P11, Y Fan, L Lv, B-C S11fi, G-Q Li aficl X-11 Wafig
`
`VACCINE TECHNOLOGY
`1390 Lentiviral Vectors with CMV or MHCII Promoters Administered /n Vivo: Immune
`Reactivity Versus Persistence of Expression
`T Kimura, RC Kaya, L Anselmi, C Sternini, H-J Wafig, B Comin-1\nduix, RM Prins,
`E Faure-Kumar, N Rozengurt, Y Cui, N Kasahara and R Stripcckc
`
`ERRATUM
`1400 Erratum to "Adhesion Receptors Medi;ite Ellicient Non-vir;il Gene Delivery"
`IS Zuhom, D Kalicharan, GT Robillard and D Hoekstra
`
`Th is ma t.e.ria I ~vas copiecl
`at the N LM a net ma y t;.e
`Subject US Co\i)yright Laws
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`original articf e ____________________________ 11_11 ·_11_11_"'_"'_· '_11 _\,_"_i,·_lY_"_' _c.,_•11_1· _1_1"_·r._,l'Y
`
`Antisense Oligonucleotide-induced Exon Skipping
`Across the Human Dystrophin Gene Transcript
`Steve D Wilton 1, Abbie M Fall 1, Penny L Harding 1, Graham McClorey 1, Catherine Coleman 1 and
`Susan Fletcher1
`
`1 Experimental Molecular Medicine Group, Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QE II Medical Centre,
`Ned/ands, Western Australia, Australia
`
`Protein-truncating mutations in the dystrophin gene
`lead to the most common childhood form of muscle
`wasting, Duchenne muscular dystrophy. Becker muscu(cid:173)
`lar dystrophy, a condition that typically arises from
`dystrophin gene lesions that do not disrupt the reading
`frame, clearly indicates that substantial domains of the
`dystrophin protein are not essential. Potential therapeu(cid:173)
`tic intervention exists during pre-mRNA splicing, where(cid:173)
`by selected exons are excised to either remove nonsense
`mutations or restore the reading frame around frame(cid:173)
`shifting mutations from the mature mRNA. Appropri(cid:173)
`ately designed antisense oligonucleotides (AOs), direc(cid:173)
`ted at amenable splicing motifs across the dystrophin
`gene transcript, block exon recognition and/or spliceo(cid:173)
`some assembly so that targeted exons are removed from
`the mature mRNA. We describe a panel of AOs designed
`to induce skipping of every exon within the human
`dystrophin gene transcript, with the exception of the
`first and last exons. Every exon targeted in vitro could be
`removed from the dystrophin mRNA, although some
`exons are more efficiently excluded than others. No
`single motif has emerged as a universal AO annealing
`site for redirection of dystrophin pre-mRNA processing,
`although the general trend is that the most efficient
`compounds are directed at motifs in the first half of the
`target exon.
`Received 24 August 2006; accepted 26 November 2006; published online
`6 February 2007. doi:10.1038/sj.mt.6300095
`
`INTRODUCTION
`Duchcnnc muscular dystrophy (DMD), the most common,
`serious form of muscular dystrophy, arises from mutations in the
`huge dystrophin gene that cause premature termination of
`translation, thereby preventing synthesis of a functional gene
`product. 1 DMD is an X-linked recessive condition occurring at a
`cited frequency of about I in 3,500 live m,rle births. Affected
`individuals show signs of muscle weakness at the ages of 3-5
`years, ,111d arc typically restricted to a wheelchair hy 12 years, as
`the absence of a functional dystrophin protein compromises
`
`muscle fiber integrity (for review sec ref. 2). llcckcr muscular
`dystrophy also arises from mutations in the dystrophin gene, hut
`in this condition clinical presentation and progression m;1y vary
`from an intcrmcdiatc/hordcrline DMD to asymptomatic This
`spectrum of severity depends 011 the quantity and quality of
`functional dystrophin produced. ( ;cncrally caused by mutations
`llcckcr musrnlar
`the reading framc;1
`that do not disrupt
`dystrophy may, in somt· cases, he diagnosed only late in lift-.
`This clearly indicates th,rt substantial segments of the prott·in,
`partirnlarly within the rod domain, arc not essential for near(cid:173)
`normal dystrophin protein function. 11'
`Following promising i11 l'itro manipulation of the dystrophi11
`tire application of a11tiscnsc
`;1go,
`prc-mllNA a few years
`remove or correct dystrophin
`to
`(AOs)
`oligonuclrntidcs
`protein-truncating mutations has rapidly progressed tow.ml
`clinical trials. 7 These trials will cm11mc1Kt' with intranrnsrnlar
`injections i11 a limited number of patients to confirm that
`promising results obtained i11 animal modclsH·'' can he achieved
`in human muscle. Even if these studies do not rt·wal any adverse
`effects and confirnr that targrtrd cxon skipping can he induced
`i11 human musck, two major issut·s must still he addrt·ssrd. The
`first challenge is to establish an clfirirnt and safl' systemic
`delivery regimen that will he infl11cnn:d by thr efficiency with
`which tht· i\O can induce t·xon skipping, a.swell as the chemistry
`employed. The scrn11d concern regards the development of
`scores of /\Os that will he required to addre.s.s the many diffcrrnt
`dystrnphin nHll,rtions that can inactivate dystrophin, the largest
`known h1111ran gene.
`llelatively few AOs will he required to restore the reading
`in a selection of DM() patients who have genomic
`frame
`the deletion hotspots. 11 '· 11
`I fowc\'l'r,
`in
`deletions clustered
`approximately one-third of ()MIJ cases arise from more subtle
`DNA changes, such as nonsense mutations, splice motif defects,
`and microinscrtions/delctions. These do not appear to he
`their rclatin:
`the dystrophin gene; hence
`clustered within
`individual incidence may rank them as hcing of only low
`priority when considering implementation of an exo11-skipping
`strategy. However, the prevalence of a mutation may 1101 ht· a
`valid parameter for prioritizing DMD cases for lrt-;1tmrnt, as
`many of these subtle, yet catastrophic I >NA changes arc likrlr to
`
`C11rrcspo11dc11c1·: Steve D Wilton, Experimental Molcrnlar Medicine Group, Centre for Ncuromuiwlar and Nc11rolo<jiwl Oimrdc11, llnivc11ity (I/ 1Vc1tcrn
`Australia, QE II Medical Centre, 4th Floor "A" /1/ock, Ned/ands, Western A111trulia 6009, A111/ru/ia. [-111ail: iwilto11(iitcyl/c•nc.11wo.cc/11.011
`
`1288
`
`Thi.s m-ater ial ,vas.copied
`atth,a NLM and m a y be
`~ul>ject USCoj'.Jcyright Laws
`
`www.moll'n1l11rtl1frll/')',OfJ! v11L 1:, 110.
`
`/ , l.~XH 12 1>ri i11ly 200 ~·
`
`..,
`
`

`

`r· -··
`?'
`
`I
`
`)
`
`I >
`
`I i.,.
`I •
`
`' /
`
`r·
`
`lliv Arnvri(,111 ~otil'ly of Ct·rw ll wr,1py
`
`Exon Skipping of All Human Dystrophin Exons
`
`be more responsive to an exon-skipping therapy. The 50 exons
`encoding the rod domain between hinges I and 4 represent the
`major portion of the protein-coding region of the dystrophin
`gene transcript. Hence, this area should contain about two-thirds
`of the subtle DNA changes as well as the major deletion hotspot
`involving exons 44-53. The removal of one or two exons from
`the rod domain would not he expected
`to compromise
`dystrophin function, as very few Becker muscular dystrophy
`patients have been identified with small deletions in the rod
`domain, and those that have been identified are generally
`asymptomatic or very mild!)' affcctcd. 12·1.l
`We describe a comprehensive list of AOs
`that
`induce
`individual exon skipping across the dystrophin gene transcript,
`excluding the first and last exons. Every cxon targeted was
`successfully removed from
`the mature dystrophin mllNA,
`although the overall efficiency varied considerably. In some
`cases, efficient excision of the target exon could not be achieved
`using a singk AO; however, a combination of two or more
`rnmpm11Hls was found to he highly effel'.live. No single splicing
`motif emerged as a reliable target to induce consistent L'Xon
`skipping, although the general lrcnd was that targeting motifs in
`the first half of an exon rL·sulted in more efficient exon skipping,
`and longn AOs (25-30-mers) lclllkd to be more effective than
`shorter counterparts.
`
`RESULTS
`four hundred seventy AOs, consisting of 2'-O-methyl modified
`bases on a phosphorothioatl' backbone (2O1\kAOs) , were
`prepared lo target motifs predicted lo be involved in processing
`the dystrophin prc-ml{NA. These rnmpounds were prepared as
`cationic lipoplcxcs and added to normal human myogenic
`rnlturcs, from which RNA was extractl'd 2-1 h after transfcclion
`and analyzed for exon skipping. Approximate!)' two thirds of the
`AOs were able to induce skipping of targeted dystrophin exons,
`hut the relative efficiencies of induced L'Xon excision varied
`rnnsidaahly. In most L"i\SL's, AOs directl'd at several different
`domains within an l'Xon could induce L'Xon skipping after cell
`tra1bfection at conccntratiom hetwL·en JOO and (,00111\I (data
`not shown). After identification of motifs found lo redirect
`drstrophin pre-ml{NA, a sernnd series of overlapping AOs was
`prepared and transkcted into rnllured cells owr a range of
`conn·ntralions. Till' more efficient AOs were reg.mkd as those
`that would induce consislrnl and sustained L'Xon skipping al
`lower t ransfi.·ct ion concentrations.
`Exon -skipping efficiL·111.:y was estimated by densitometry
`analrsis of gel images comparing induced shortened dystrophin
`i11
`mRNAs to the intact lramnipt aflL·r
`,·itro transfi.·ction at
`100 nl\1. Equal amounts of till' induced and i11t.1ct transcripts
`would be regarded as repre.sl'llting SO'! ;, efficienq·, whneas ,lll
`estimate of 2'.1% cxon skipping would he represenlt·d hr till'
`intact transcript prod11cl being thrl'l' times nlorL' .1hund.t11l th,t11
`thl' hand reprt·\enting thL· induced transcript.
`\Ve haw arhitraril)' tla\\ilied the d)'\lrnphi11 nuns a\ Trpcs I,
`2, J, or •I, h,l\Cd 11pon thl' k\'cl ,1nd 1t1,1nm·r in whkh the non
`ct11 ht· l'XLi\cd fro111 lhl' n1.1tun· 111l{N,\ . Type, I, 2. and ,I rcfl-r lo
`l'XOn\ that can lie n·mm·nl with high (gn·.1tn th.111 _l(l'!i, H·l.1tive
`to tla· inta<.:t tran,nipt) , n1edium (IH'l\\'l'e11 Ill and .111%), and
`
`low (less than JO%) levels of exon-skipping efficiency, respec(cid:173)
`tively. Type 4 exons include those from which additional flanking
`exons are also removed and/or more than one AO was required
`for efficient exon skipping.
`Nucleotide sequences of the AOs developed to remove the
`dystrophin exons are described in Table I. Relative annealing
`coordinates within each dystrophin exon arc shown in Figure 1,
`with details of the exon and intron length. Representative gel
`images illustrating targeted dystrophin exon skipping are shown
`in Figure 2a (exons 35 and 53) for Type 1 exons, Figure 2b
`(cxo ns 11 and 21) for Type 2 exons, Figure 2c (cxons 26 and 76)
`for Type 3 exons, and Figure 2d (exons 20 and 34) for Type 4
`exons.
`The majority of exons classified as Types 1 and 2 arc found in
`the first two-thirds of the dystrophin gene transcript, possibly
`reflecting greater effort expended in optimizing exon skipping in
`this region. Most Type 3 exons occur in the second half of the
`dystrophin gene, and these arc yet to be fully optimized.
`Type •I dystrophin exons may he regarded as a miscellaneous
`group. The induction of shortened transcripts generally occurs al
`substantial levels, typically greater than 200,,'c, relative to induction
`of the intact transcript. 1 lowever, excision of these particular
`exons from the mat11re mRNA differs from that of Types 1-3 in
`that either a combination of AOs is required or the target exon
`excision includes additional flanking exons. For example, a single
`AO induced strong and consistent cxon 8 removal; however, all
`induced transcripts were also missing exon 9. This effect has also
`been observed in the canine dystrophin gene transcript 1-1 and, as
`there was no substantial sequence homology between the exon 8
`AO and exon 9, this was interpreted as indicating closely linked
`processing of these two exons. It was interesting to note that
`skipping of only exon 9 was induced with an AO directed at that
`target, suggesting some order or direction to dystro phin pre(cid:173)
`mRNA processing in this region. 'fargeting exon 17 showed a
`similar pattern. All induced transcripts skipped both cxons 17
`and 18, with no transcripts missing only exon 17 detected.
`I )irecting J\Os at cxon 18 resulted in precise remov.il of the target
`L'xon (data not shown).
`Dystrophin L'xo n 10 was classified as another T)•pe •I exon as
`this exon could not he specifically removed h)' any single AO.
`Rather, several shortened transcripts missing multiple cxons
`( 10- 11, 10-12, 9-1:1, and 9-15) were detL'Cted, hut
`these
`alternative!)' processed transcripts wne not generated in a
`consistent manner {data not shown). l lowever, the combination
`of two A Os, 111 0t\( -- OS ·I· I<,) and 111 0i\( I 98 I 119), induced
`rqiroducihlc L'Xon IO skipping in a dose-depe1Hk·nt mannL'I', as
`WL'II as consistent!)' inducing an in-frame transcript n1issing
`L'Xon.s 9-1.1. ·1:1rgeting L'Xllll
`'.1·1 with a singk AO
`induced
`transcripts missing L'Xlln 5,1 or exons S-1 and 55 in approximate!)'
`equal amounts (data not shown).
`The majorit)' of /\Os designed to inducL' non 20 skipping
`WL're found to ht· ineffrctil'l' or to indun· \'LT)' low levels of
`targeted t·xo11 skipping, even aftn transfrction at conCL'nlralions
`as high as c,OO 111\I (data not shown). I lmvewr, thc combination
`ol 1l20A( I ·1·1 I 71) and l120A( I 1•17 I !Ml) w,1,s very efficient
`in bringing about exon 20 exclusion (Figure 2d). /\ single AO,
`112.0A( / .19 / (,(J), was suhsL·quentl)' developed that
`induced
`
`,\f,,l,•m/11r '/'/11·rnt•Y \'Ill. l:, 1111 , 7. julr 2007
`
`Th is m ate r ial w a:scop.ied
`att.he NLM and may be
`5'1.Jb-ject useo,µ.yright Laws
`
`1289
`
`

`

`Exon Skipping of All Human Dystrophin Exons
`
`Table 1 Nucleotide sequences, annealing coordinates, and target motif of AOs designed to remove dystrophin cxons 2-78, classified according to
`exon skipping efficacy
`Maximum ESEfinder values over threshold
`
`Exon AO name
`Type I
`
`5' sequence
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`9
`
`12
`
`13
`
`14
`
`SF2/ASF
`
`SC35
`
`SRp40
`
`SRp55 Donor I acceptor
`
`4.2 (2)
`
`2.7
`
`3.0
`
`4.1
`
`2.3
`
`2.0
`
`4.0 (2)
`
`5.3 (2)
`
`3.8
`
`3.8 (2)
`
`3.0
`
`3.3 (3)
`
`5.7
`
`5.4
`
`2.7 (2)
`
`2.(,
`
`3.1 (2)
`
`3A
`
`3.2 (2)
`
`5.2 (2)
`
`3.15
`3.9 (3)
`
`4.2 (2)
`
`5.3 (2)
`
`3.7
`
`3.1
`
`.l.'J (,I)
`
`3.1
`
`3.0
`
`.1.3
`
`.\.0
`
`ESE
`ESE
`ESE
`ESE
`ESE
`ESE
`
`A/ESE
`
`ESE
`
`ESE
`
`ESI:
`
`ESE
`
`15
`
`16
`
`18
`
`19
`
`22
`
`23
`
`24
`
`25
`
`27
`
`28
`
`29
`
`•1.0
`
`J.I (2)
`
`.I.I (.1)
`
`2.8 (.1)
`
`2.2 (2)
`
`2.0
`
`2.8
`D
`2.7 (2)
`
`-1.(,(2)
`
`2.'i
`
`2.">
`
`2.11
`
`2.-1
`
`2.(,
`
`.l.7
`
`2.7
`
`.\.2 (2)
`
`2.5
`
`J.3 (2)
`
`J.2 (2)
`
`.U
`
`J .. l
`
`•1.1 (2)
`
`5.5 LI)
`
`2.8
`
`3.'J (2)
`
`.\.0
`
`.1.0
`
`.u
`.1.(, (2)
`
`.\.8
`
`.U
`
`2.8
`
`2.'i
`
`A/1:SE
`
`ESE
`
`ESE
`
`ESE
`
`ESE
`
`ESI:
`
`ESE
`
`ESE
`
`ESE
`
`ES!:
`
`H2A(+l2+41)
`H3A(+20+40)
`H4A(+ll+40)
`HSA(+25+55)
`H6A( +69+91)
`H7 A( +45+67)
`H9A(-06+23)
`Hl2A(+52+75)
`Hl3A(+77+100)
`Hl4A(+32+61)
`1115A(+48+71)
`ll16A(-12+19)
`II l 8A( +24+53)
`HMl9A(t35+65)
`1122A( + 125+ 146)
`H23A ( +69+98)
`H24A(+51+73)
`H25A(+95+119)
`1127A(+82+106)
`H28A ( +99+ 124)
`ll29A(+57+81)
`H30A( +25+50)
`H3ID(+03-22)
`H32A(44+73)
`H33A(+64+88)
`H35A( +24+53)
`1137A(+l34+157)
`H38A(+88+112)
`ll39A(+62+91)
`H41A(+44+69)
`ll42A(-4+23)
`I 147A(-06t24)
`ll49A(-11+16)
`115 IA( +66+90)
`ll52A(+l2+41)
`ll53A( +39+69)
`1 l72A( +02+28)
`ll74A( +48 t 72)
`I 175A( +34+58)
`1l77A(+l6+42)
`1178"( HJ.1 +2'1)
`
`CCA UUU UGU GAA UGU UUU CUU UUG AAC AUC
`GUA GGU CAC UGA AGA GGU UCU
`UGU UCA GGG CAU GAA CUC UUG UGG AUC CUU
`UCA GUU UAU GAU UUC CAU CUA CGA UGU CAG U
`UAC GAG UUG AUU GUC GGA CCC AG
`UGC AUG UUC CAG UCG UUG UGU GG
`CCC UGU GCU AGA CUG ACC GUG AUC UGC AG
`UCU UCU GUU UUU GUU AGC CAG UCA
`CAG CAG UUG CGU GAU CUC CAC UA<;
`GUA AAA GAA CCC AGC G(;U CUU CUG UCC AUC
`ucu UUA AAG CCA <;uu c;uc; uc;A AUC
`CUA <;Au CC(; CUU UUA AAA CCU GUlJ AAA ACA A
`CAG cuu cue; AGC GA(; UAA ucc AGC uc;u (;AA
`c;cc UGA c;cu c;Au cue; cue; c;cA ucu uc;c Ac;u u
`CUG CAA uuc co; (;Ac; ucu cue; C
`CG(; CUA AUU UCA (;Ac; c;c;c GCU uuc uuc c;Ac
`CAA c;c;c; CAG c;cc AUU CCU CCU UC
`uuc; AGU ucu GUC UCA AGU CUC c;AA c;
`UUA AGC CCU cuu GUG CUA CA(; c;uc; c;
`CAG AGA uuu CCU CAG cue cc;c CAG (;A
`ucc CCC AUC UGU U,\G cc;u cue; uc;c C
`ucc UGG GCA GAC ucc; AUG CUC uc;u UC
`UA(; UUU CUG AAA UAA CAU AUA CCU c;
`cuu CUA CAC c;cu GCU CAA AAU UG(; cue; c;uu
`CCG UCU GCU UUU UCU GUA CAA UCU c;
`ucu GUG AUA CUC uuc AG(; uc;c ACC uuc uc;u
`uuc UGU CUG MA uc;c; cue; CAA AUC
`UGA AGU CUU CCU CUU UCA GAU lJCA C
`uuu CCU CUC c;cu uuc uc:u CAU cue; uc;A uuc
`CAA GCC CUC AGC uuc; CCU ,\C(; CAC: uc;
`AUC GUU ucu UCA cc;c; ACA ca)(; uc;c: uc;c;
`CA(; c;c;c; CAA CUC uuc CAC CA(; UAA cue; ,\,\A
`cue; CUA uuu CA(; uuu CCU c;c;c; c;t\A AM;
`ACA UCA A(;(; AA<; AU(; c;C,\ uuu CUA c;
`ucc MC uc;c; c;c;A cc;c cue uc;u ucc: AM ucc
`CAU UCA ACU cuu GC:C uc:c c;c;u ucu {;J\A G(;u c;
`c;uc; U(;,\ AAG cue; ,\(;c; GGA CGA GGC AGC
`C(;A c;(;c lJ(;(; CUC,\(;(; c;c;c; c;A(; ucc u
`c;c;A CA(; GCC uuu AU(; uuc GU(; cue; C
`cu<; uc;c uuc; uc;u ccu c;c;<; c;M; c;Ac UCA
`ucu CAU uc;c; c:uu ucc AG(; c;(;U AUU UC
`
`49
`
`51
`
`52
`
`53
`
`72
`
`74
`
`75
`
`77
`
`78
`
`'[}pr 2
`
`II
`
`21
`
`Ill IA(+7St97)
`1121A( -186 t-108)
`
`CALI C:UU CU<; ,\UA AUU UUC CUC; lJlJ
`( ;uc ll( ;c AUC: CA(; ( ;A,\ C:AlJ ( ;( ;c; !JC
`
`1290
`
`This mat e,rial ,,ras co,p,ied
`at the N LM and ma y be
`Su bject US Cop yright La ws
`
`J,5
`
`4.8 (.1)
`
`3 .. 1
`
`),6
`
`3.2
`
`J.I (2)
`(1.6
`
`3,6
`
`3.5
`
`6.0 (2)
`
`3.8 (2)
`
`3.0 (3)
`
`2.7 (3)
`
`3.1
`
`2.8
`
`5.2 (3)
`
`3A (2)
`
`4.7 (5)
`
`4,0 (2)
`
`3.3 (2)
`
`),8 (2)
`
`·1.0 (2)
`
`ESE
`
`ESE
`
`LSE
`
`ESE
`
`LSE
`
`ESE
`
`J:SE
`
`ES!'
`
`!'SL
`
`.1,9 (2)
`
`•1.8 (.1)
`
`_J.,J
`
`ES!
`·l.·1
`f.1i>le I i\ co11lill11ed 011 1hr lollowirHJ p.1q,•
`
`ll'll'll'.lllli/,•rnfllr//11·mt•)'.MJ.: I'll!. I:, 1111. 7, jul)' 21lll7
`
`30
`
`31
`
`32
`
`33
`
`35
`
`37
`
`38
`
`39
`
`41
`
`42
`
`47
`
`3.1
`•U (2)
`
`5.6
`
`2.8
`5.6
`
`J. I (·I)
`
`3..1 (.\)
`
`,1,2
`
`·l.2 (.I)
`
`.1. 1)
`
`.1.7 (2)
`
`·1.3
`
`2.7
`
`.1.6
`
`2.">
`.1,(,
`
`2.5
`
`2.7
`
`J.8
`
`5.1
`
`2.'J(.l)
`
`,1.(, (2)
`
`3.'J (2)
`
`] ,(,
`
`•1.2 (2)
`
`3.7
`
`2.'J
`0(2)
`
`.1.1
`
`2.'J
`:>.8
`
`2.'J (2)
`
`4,4 (2)
`
`2.7
`
`5.H
`3.<,
`
`PiE
`
`I>
`
`ES!:
`
`ESE
`
`ESE
`
`ESE
`
`ESE
`
`ESE
`
`ESE
`
`ESE
`
`A
`
`A/ESE
`
`

`

`' 1/w A11H·1ic,111 \ocil'ty of Ct'llt' llwr,1py
`
`Exon Skipping of All Human Dystrophin Exons
`
`Table 1 Continued
`
`Maximum ESEfinder values over threshold
`
`Exon AO name
`
`5' sequence
`
`SF2/ASF
`
`SC35
`
`SRp40
`
`SRp55 Donor/acceptor
`
`36
`
`.JO
`
`43
`
`4.J
`
`46
`
`48
`
`57
`
`60
`
`61
`
`ll36A(+22+51)
`
`UGU GAU GUG GUC CAC AUU CUG GUC AAA AGU
`CUU UGA GAC CUC AAA UCC UGU U
`H•IOA(-5+17)
`H-IJA(+IOl+l20) GGA GAG AGC UUC CUG UAG CU
`UGU UCA GCU UCU GUU AGC CAC UGA
`114.JA( +61 +8-1)
`ll46A(+l07+137) CAA GCU UUU CUU UUA GUU GCU GCU CUU UUC C
`uuc UCA c;c;u AAA GCU CUG GAA ACC UGA AAG
`ll48A(-07+23)
`CUG GCU uc:c AAA uc;c; GAC CUG AAA MG AI\C
`CUG c;c:c; A(;C AI\G GUC cuu c;Ac GUG GCU CAC
`c;c;c; cuu C,\U c;C,\ GCU c;cc uc;A CUC GGU CCU C
`C:AU (L\ c;uc: UA(; c;M (;A(; c;c;c: CGC: uuc:
`
`IIS7A (-12+18)
`
`3.2 (2)
`
`3.4 (3)
`
`4.1 (2)
`
`3.5
`
`3.0
`
`3.6 (3)
`
`3.0 (2)
`
`3.4
`
`3.2
`
`5.6 (2)
`
`4.4 (2)
`
`3.5
`
`2.8
`
`2.8 (2)
`
`4.0 (2)
`
`5.