www.rnajourna|.org
`
`exon ski
`
`—-
`
`_
`
`
`
`,
`Review of 21:?“ ~55?
`l
`i
`‘
`PP i_
`‘ ,.
`Review of the birth of new exons
`
`Improved amber and opal
`
`suppressor tRNAs
`
`Cloning and expression analysis
`
`of piRNA-like RNAs
`
`"
`
`Modulation of group I intron
`catalysis by a peripheral metal ion a
`
`Sarepta Exhibit 1057, Page 1 of 20
`
`

`

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`

`

`A PUBLI CAT I ON OF THE R
`VOL . 1 3, NO. 1 0 f!1tJi;\ OCTO BER 2007
`~
`
`CONTENTS
`
`Mini-Review
`The birth of new exons: Mechanisms and evolutionary consequences
`Rotem Sorek
`
`Review
`Antisense-mediatcd exon skipping: A versatile tool w ith therapeutic and research
`applications
`Annemieke Aartsma-lfos and Gert-Jan B. van Ommen
`
`Letter to the Editor
`The tolerance to exchanges of the Watson- Crick base pair in the hammerhead ribozyme
`core is determined by surrounding elements
`Rita Przybilski and Christian Hammann
`
`Bioinformatics
`Effect of target secondary structure on RNAi efficiency
`Yu Shao, Chi Yu Cha,,, A11i/ Maliyekke/, Charles E. Lawrence, Igor B. Roni11son, and Ye Ding
`
`Reports
`The Saccharomyces cerevisiae Pus2 protei n encoded by YGL063w ORF is a mitochondrial
`tRNA:'V2 7 /28-synthase
`Isabelle Behm-Ansmant, Christiane Bra11la11t, and Yuri Motorin
`
`Specific binding of a Pop6/Pop7 heterodimer to the P3 stem of the yeast RNasc MRP and
`RNase P RNAs
`Anna Perederina, Olga Esakova, Hasan Koc, Mark E. Schmitt, and Andrey S. Krasil11ikov
`
`Articles
`Modulation of individual steps in group I intron catalysis by a peripheral meta l ion
`Marcello Forconi, Joseph A. Piccirilli, and Daniel Herschlag
`
`1603
`
`1609 OA
`
`1625
`
`163 1
`
`1641
`
`1648
`
`1656
`
`(co111i1111ed)
`
`Cover Illustration: Crystal structure of a phage Twort group I ribozyme-product complex (PDB code: ly0q; Golden, B.L.,
`Kim, H., and Chase, E. 2005. Crystal structure of a phage Twort group I ribozyme product complex. Nat. Struct. Mo!. Biol.
`12: 82-89). Image details: ribozyme derived from the second group I intron in the orfl42 gene (orf142-12): ribbon-plate
`red, P3-P7 region-green, P4-P6 domain- blue, P9-P9. / domain-purple,
`representation, transparent surface, P 1-P2 domain-
`Pl. J- P7.2 subdomain- yellow, nucleotides not included in these domains-white; oligonucleotide representing a 5' exon:
`ball-and-stick representation, cyan. The image was generated with the Accelrys Discovery Studio Visualizer. Cover image
`provided by the Jena Library of Biological Macro111olewles (JenaLib; www.Jl.i-leibniz.de/ TMA GE. lrtml).
`
`Published by Cold Spring Harbor Laboratory Press
`
`

`

`Contents (continued)
`
`Systematic analysis of microRNA expression of RNA extracted from fresh frozen and
`formalin-fixed paraffin-embedded samples
`Yaguang Xi, Go Nakajima, Elaine Gavin, Chris G. Morris, Kenji Kudo, Kazuhiko Hayashi, and Jingfang Ju
`
`Nuclear factors are involved in hepatitis C virus RNA replication
`Olaf Isken, Martina Baroth, Claus W. Grassmann, Susan Weinlich, Dirk H. Ostareck, Antje Ostareck-Lederer,
`and Sven-Erik Behrens
`
`Cloning and expression profiling of testis-expressed piRNA-like RNAs
`Seungil Ro, Chanjae Park, Rui Song, Dan Nguyen, Jingling Jin, Kenton M. Sanders, John R. McCarrey,
`and Wei Yan
`
`Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in
`vivo. Part 1: Minimizing misacylation
`Erik A. Rodriguez, Henry A. Lester, and Dennis A. Dougherty
`
`Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in
`vivo. Part 2: Evaluating suppression efficiency
`Erik A. Rodriguez, Henry A. Lester, and Dennis A. Dougherty
`
`A functional interaction of SmpB with tmRNA for determination of the resuming point
`of trans-translation
`Takayuki Konno, Daisuke Kurita, Kazuma Takada, Akira Muto, and Hyouta Himeno
`
`Alternative splicing of the ADAR1 transcript in a region that functions either as a 5' -UTR
`or an ORF
`S0ren Lykke-Andersen, Serafi'n Pinal-Roma, and Jergen Kjems
`
`Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in
`mammalian cells
`Ewa Grudzien-Noga/ska, Jacek Jemielity, Joanna Kawa/ska, Edward Darzynkiewicz, and Robert E. Rhoads
`
`Polyadenylation site choice in yeast is affected by competition between Npl3 and
`polyadenylation factor CFI
`Miriam E. Bucheli, Xiaoyuan He, Craig D. Kaplan, Claire L. Moore, and Stephen Buratowski
`
`Defining the optimal parameters for hairpin-based knockdown constructs
`Leiming Li, Xiaoyu Lin, Anastasia Khvorova, Stephen W. Fesik, and Yu Shen
`
`Methods
`Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells
`Chyi-Ying A. Chen, Yukiko Yamashita, Tsung-Cheng Chang, Akio Yamashita, Wenmiao Zhu,
`Zhenping Zhong, and Ann-Bin Shyu
`
`A novel monoclonal antibody against human Argonaute proteins reveals unexpected
`characteristics of miRNAs in human blood cells
`Peter T. Nelson, Mariangels De Planell-Saguer, Stella Lamprinaki, Marianthi Kiriakidou, Paul Zhang,
`Una O'Doherty, and Z issimos Mourelatos
`
`Instrumentation and metrology for single RNA counting in biological complexes or
`nanoparticles by a single-molecule dual-view system
`Hui Zhang, Dan Shu, Faqing Huang, and Peixuan Guo
`
`A simple array platform for microRNA analysis and its application in mouse tissues
`Xiaoqing Tang, Jozsef Gal, Xun Zhuang, Wangxia Wang, Haining Zhu, and Guiliang Tang
`
`RNA: Instructions for contributors
`
`OAOpen Access paper
`
`1668
`
`1675
`
`1693
`
`1703
`
`1715
`
`1723
`
`1732
`
`1745
`
`1756
`
`1765 OA
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`1775
`
`1787
`
`1793
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`1803
`
`1823
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`REVIEW
`
`Antisense-mediated exon skipping: A versatile tool
`with therapeutic and research applications
`
`ANNEMIEKE AARTSMA-RUS and GERT-JAN B. VAN OMMEN
`DMD genetic therapy group, Department of Human Genetics, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands
`
`ABSTRACT
`
`Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able
`to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of
`alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted
`reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would
`convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most
`promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the
`applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous
`exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In
`addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This
`knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon
`skipping approach.
`
`Keywords: exon skipping; splicing; Duchenne muscular dystrophy; antisense oligonucleotides; therapy
`
`INTRODUCTION
`
`Antisense oligonucleotides (AONs) are mostly known for
`their ability to hybridize to a sense target sequence, which
`leads to RNase H cleaving of the RNA:DNA hybrid and
`results in specific gene expression knockdown (Hausen and
`Stein l 970; Zamecnik and Stephenson l 978). This ap(cid:173)
`proach offered useful opportunities to study development
`because it allowed timed gene knockdown in early or later
`stages of development, as well as therapeutic opportunities
`to knockdown genes involved in cancer, inflammatory
`diseases, and viral infections. Currently, an AON to treat
`CMV-induced retinitis (Vitravene) has been registered as a
`drug, and other AONs to treat cancer and inflammatory
`diseases are in phase II and III clinical trails (Marwick 1998;
`Kurreck 2003). However, with the emergence of RNAi,
`which turned out to be a more efficient and more predict(cid:173)
`able tool for expression knockdown, the field of AON(cid:173)
`induced knockdown has gone in decline (Elbashir et al.
`2001 ). A notable exception is the modulation of pre-mRNA
`
`splicing to induce exon skipping, where RNase H-indepen(cid:173)
`dent AONs are employed to block splicing signals (Kole
`and Sazani 2001). This approach has gained increasing
`interest over the past decade (van Deutekom and van
`Ommen 2003). Actually, antisense-mediated exon skipping
`is currently one of the most promising therapeutic
`approaches for Duchenne muscular dystrophy (DMD). A
`first-in-man trial has recently been completed successfully
`in our institute (J.C.T. van Deutekom, A.A.M. Janson, I.B.
`Ginjaar, W.S. Frankhuizen, A. Aartsma-Rus, M. Bremmwe(cid:173)
`Bout, J.T. den Dunnen, K. Koop, A.J. van der Kooi, N.M.
`Goemans, et al., in prep.) and a second trial is about to start
`in the United Kingdom (Muntoni et al. 2005; F. Muntoni,
`pers. comm.). This review describes the mechanism of
`antisense-mediated exon skipping for DMD and gives an
`overview of other exon skipping applications reported thus
`far. It discusses how the numerous AONs designed for
`DMD exon skipping give us insight into splicing of the
`DMD gene in particular, but splicing in general as well.
`Finally, ways to implement exon skipping in future appli(cid:173)
`cations will be discussed.
`
`Reprint requests to: Annemieke Aartsma-Rus, DMD genetic therapy
`group, Department of Human Genetics, Leiden University Medical Center,
`P.O. Box 9600, 2300 RC, Leiden, The Netherlands; email: a.111.rus@
`lumc.nl; fax: 31-71-5268285.
`Article published online ahead of print. Article and publication date are
`at http://www.rnajournal.org/cgi/doi/lO. I 261/rna.653607.
`
`DUCHENNE MUSCULAR DYSTROPHY
`
`Duchenne muscular dystrophy is a severely invalidating,
`progressive neuromuscular disorder (Emery 2002). Patients
`
`RNA (2007), 13:1609-1624. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 RNA Society.
`
`1609
`
`

`

`Aartsma-Rus and van Ommen
`
`are wheelchair bound before the age of twelve, o ften require
`assisted ventilatio n later in life, and generaUy die in their
`early twenties. T he disease is caused by mutations in the
`DMD gene that abolish the production of functional
`dystrophin (Ho ffman et al. 1987). This protein consists
`of two essential functio nal domains connected by a central
`rod domain that is partly d ispensable (Hoffman et al. 1988;
`Koenig et al. 1988). Dystrophin links the cytoskeleto n to
`the extracellular matrix and is thought to be req uired to
`m aintain muscle fiber stability d uring contractio n (Matsu(cid:173)
`m u ra and Campbell 1994). M utations tha t d isrupt the open
`reading frame result in prematu rely truncated pro teins
`unable to fulfi U their b ridge functio n (Fig. I). Ultimately,
`this leads to muscle fiber damage and the conti nuous loss
`
`Dystrophin Protein
`
`,■-~-Ce•n-lra.lrod-doma-in--. . ~
`
`Aclin-blnding domain
`
`IH!ystrogiycan binding domain
`
`.#J.i,;.f~
`
`DMD deletion exon 48-50
`
`➔#J./eef!► lntton 48/lntron 50 4#0!,;.i- lntron 52
`! Open reading frame disrupted
`1¥0J,El;-.,4 iai,i-iMii•i,1-t♦
`Premarura slOp codon
`! Non functional dystrophin
`
`• Reading frame restoration by exon 51 skipping
`
`AON
`➔#t-i,();- lntron 48/lntron 50 4#(,1,W.i- lntron 52 $#J.i,i-f---
`! Exon 51 ~kipped _. Open reading frame restored
`1#1-1,£!:M#J.l,f.t►
`! Partly functional dystrophin
`
`•
`
`FIGURE 1. Antisense-mediated exon skipping for Duchenne mus(cid:173)
`cular dystrophy. The dystrophin protein (11pper panel) contains an N(cid:173)
`terminal actin-binding domain connected to a ~-dystroglycan binding
`domain by the central rod domain. Dystroglycan is a lransmembrane
`protein that is bound to the extracellular protein laminin-2. Dystro(cid:173)
`phin thus fulfills a bridge function in muscle fibers by linking the
`cytoskeletal actin to the extracellular matrix. In Duchenne muscular
`dystrophy (middle panel), the open reading frame is disrupted (in this
`example by a deletion of exons 48-50, the most common mutation in
`DMD patients ), resulting in a premature stop codon and a truncated
`dystrophin, which is unable to fulfill its bridge function. Antisense
`oligoribonucleotides (AONs) can be employed to restore the open
`reading frame (lower panel). Specific AONs hybridize to exon 51 and
`hide this exon from the splicing machinery, resulting in the splicing of
`exon 51 with its flanking intron. This restores the open reading frame,
`allowing the generation of an internally deleted dystrophin, that
`contains both the actin- and dystroglycan binding domains and
`therefore is partially to largely functional.
`
`1610
`
`RNA, Vol. 13, No. 10
`
`of muscle fibers, replacement of muscle tissue by fat and
`fibrotic tissue, impai red muscle function, and eventually
`the severe phenotype observed for DMD patients.
`In contrast, mutatio ns that maintain the o pe n read ing
`frame allow for the generation of internally deleted, but
`pa rtia lly functional dystrophins (Monaco et al. 1988). These
`muta tions are associated with Becker m uscular dystrophy
`(BMD), a much milder disease when compared to DMD.
`Patients generally remain ambulant until la ter in life and
`have near n ormal expectancies, although more severely
`affected patients have been reported as well (Emery 2002).
`T he DMD gene is the largest known huma n gene and its
`79 exons span an asto nishing 2.4 Mb (Roberts et al. 1993,
`1994). Over 70% of all DMD and BMD patients suffer from
`deletio ns of one or mul tiple exons (Aartsma-Rus et al.
`2006c). Mildly affected BMD patients carrying deletions
`that involve over two thirds of the central rod doma in have
`been described, suggesting that this do main
`is
`largely
`dispe nsable. Dyst rophin can be largely functio nal as lo ng
`as the N- a nd C-terminal domains a re p resent to convey
`the link between the cytoskeleto n a nd the extraceUular
`matrix (Engla nd et al. 1990; Mirabella et a l. 1998). D ue to
`this rather unique featu re, the counterintuitive skipping of
`addi tional internal exons can be employed to enlarge a
`deletio n, but at the same time restore the o pen reading
`fram e and thus convert a severe DMD into a milder BMD
`phenotype (Fig. I; van Dcutcko m ct a l. 2001 ).
`
`THE EXON SKIPPING APPROACH
`
`Antisense-mediated modulation of pre-mRNA splicing has
`been pioneered by Rysza rd Kole (Dominski a nd Kole
`1993) . In the first experiments, AONs were ai med at
`activated cryptic splice sites in the 13-globin (HBB) and
`fi brosis
`tra nsmem bran e conducta nce
`regulator
`cystic
`(CFTR) genes in o rder to restore normal splicing in 13-
`thalassemia a nd cystic fib rosis patients (Dominski and Kole
`1993; Sierakowska et al. 1996; Friedman et al. 1999). Even
`tho ugh this approach does no t technically qualify as exon
`skipping (but rather the redirectio n o f normal sp licing), it
`does o ffer therapeutic potential for diseases where m uta(cid:173)
`tions often induce cryptic splice sites such as the Hutch(cid:173)
`inson-Gil fo rd progeria syndrome (Scaffid i and Misteli
`2005). In fact, for most genetic disorders an estimated
`5%- L0o/o o f mutatio ns induce abnormal splicing ( Krawczak
`et al. 1992; Cartegni et al. 2002), pa rt of which can, in
`principle, be corrected .
`A finding of the group of Matsuo eventually alerted the
`DMD field to a potential therapeutic application of exon
`skipping for DMD. Matsuo and colleagues observed that a
`52-base pair (bp) deletio n within exon 19 resulted in the
`skipping of this exon in the so-called DMD Kobe patient
`(Matsuo et al. 1990, 1991 ). This hinted at th e presence of
`a motif wi thin this 52-bp deletion required for proper
`inclusion of exon 19 in the mRNA. Indeed , AONs ta rgeting
`
`

`

`part of this deletion induced exon 19 skipping in vitro and
`in human control lymphoblastoma cells (Takeshima et al.
`1995; Pramono et al. 1996).
`The feasibility of the approach was then studied in
`parallel in patient-derived cell lines and in cells from the
`mdx mouse model. This mouse carries a nonsense point
`mutation in the in-frame exon 23 (Sicinski et al. 1989).
`Thus, by skipping exon 23 the nonsense mutation is
`bypassed while the reading frame is maintained. Proof of
`principle on RNA level was obtained first in cultured
`muscle cells from the mdx mouse by two groups indepen(cid:173)
`dently (Dunckley et al. 1998; Wilton et al. 1999). In both
`cases, the reading frame was restored on RNA level as
`analyzed by RT-PCR analysis. Our group was the first to
`show restoration of dystrophin on protein level after
`in cultured muscle cells
`targeted exon 46 skipping
`from two DMD patients with an exon 45 deletion (van
`Deutekom et al. 2001 ). The wide therapeutic applicability
`was then confirmed by others and us in numerous patient(cid:173)
`derived cell cultures (Takeshima et al. 2001; Aartsma-Rus
`et al. 2003, 2004a; Surono et al. 2004; Aartsma-Rus et al.
`2007). The majority of these mutations involved deletions
`of one or more exons, but reading frame restoration for
`nonsense point mutations and single exon duplications has
`been reported as welJ. Notably, for single exon duplications,
`skipping either one of the duplicated exons will restore the
`wild-type transcript and dystrophin protein (Aartsma-Rus
`et al. 2007). Some mutations require the skipping of two
`exons in order to restore the reading frame. We confirmed
`that this so-called double exon skipping is indeed feasible
`using a combination of individual AONs targeting the two
`different exons (Aartsma-Rus et al. 2004a). Remarkably,
`the efficiency of this double exon skipping approach was
`only slightly lower than that of single exon skipping
`(~70%- 75% versus 750/<r80% dystrophin-positive myotubes,
`respectively). In parallel, results in mdx mouse underlined
`the therapeutic promise of exon skipping (Mann et al.
`2001, 2002; Lu et al. 2003). Local intramuscular injections
`of an optimized AON resulted in ~20% of wild-type
`dystrophin levels accompanied by improvement in muscle
`histology and function (Lu et al. 2003). Dystrophin protein
`was detectable by Western blot analysis for at least 3
`months after a single intramuscular injection.
`In theory, exon skipping would be applicable to the
`majority of DMD patients. Exceptions are mutations
`located between exon 64 and exon 70, which are essential
`for protein function, deletions that abolish alJ actin-binding
`sites in the N-terminal region or involve the first or the last
`exon, and large chromosomal rearrangements such as
`translocations. These mutations are uncommon and make
`up less than 10% of all mutations (Aartsma-Rus et al.
`2006c). Thus exon skipping can theoretically be applicable
`for up to 90% of DMD patients (Aartsma-Rus et al. 2004a).
`A disadvantage of the AON approach is that it is
`mutation specific in that different mutations require the
`
`Antisense-mediated exon skipping
`
`skipping of different exons to restore the open reading
`frame. Fortunately, DMD deletions and duplications
`mainly occur in two hot spot regions, i.e., the major hot
`spot region (involving exon 45 to exon 53) and the minor
`hot spot region (located between exon 2 and exon 20)
`(Liechti-Gallati et al. 1989; Beggs et al. 1990; White et al.
`2006). Therefore, by strategically choosing target exons,
`through the skipping of eight different exons, this strategy
`would be therapeutic for over 50% of alJ patients (van
`Deutekom and van Ommen 2003; Aartsma-Rus and van
`Deutekom 2007). The most notable example is exon 51
`skipping, which is applicable to almost 25% of DMD
`patients with a deletion, or 16% of all DMD patients
`(Aartsma-Rus and van Deutekom 2007).
`To obtain proof of concept in humans, a "first-in-man
`study" on exon skipping was undertaken by our center in
`collaboration with Prosensa B.V. using 2' -O-methyl phos(cid:173)
`phorothioate AONs (chemistries will be discussed in more
`detail later) (J.C.T. van Deutekom, A.A.M. Janson, I.B.
`Ginjaar, W.S. Frankhuizen, A. Aartsma-Rus, M. Bremmwe(cid:173)
`Bout, J.T. den Dunnen, K. Koop, A.J. van der Kooi, N.M.
`Goemans, et al., in prep.). Four DMD patients received a
`single, local intramuscular injection with AONs targeting
`exon 51 and a biopsy was taken one month later. Pre(cid:173)
`liminary results are very promising and no serious adverse
`effects were observed or reported by the patients as a result
`of AON injection. Another local study using morpholino
`AONs is to start soon in the United Kingdom. These first(cid:173)
`in-man studies are an important step toward the clinical
`application of antisense-mediated exon skipping for DMD.
`A systemic pilot study has been performed by Takeshima
`and colleagues in a single DMD patient at a very low dosage
`using phosphorothioate RNA (0.5 mg/kg) (Takeshima et al.
`2006).
`
`AON DESIGN AND MODE OF ACTION
`
`The first targets to induce exon skipping are the donor and
`acceptor splice sites and the branch point sequence. These
`sites have indeed been successfully targeted in the majority
`of the exon skip applications, including exon skipping
`for DMD (Table l; Dunckley et al. 1998; Mann et al. 2002;
`Wilton and Fletcher 2005). However, they consist of con(cid:173)
`sensus sequences shared with many different genes and
`consequently targeting them involves the risk of mistarget(cid:173)
`ing splice sites of other genes. Alternatively, it has now been
`shown that exon skipping can be induced by targeting
`exon-internal sites, which has been successful in the DMD
`and WTI genes (van Deutekom et al. 2001; Renshaw et al.
`2004; Aartsma-Rus et al. 2005; Wilton and Fletcher 2005).
`Proper recognition by the splicing machinery and inclusion
`into the mRNA is thought to depend on exonic splicing
`enhancer (ESE) motifs for the majority of exons (Cartegni
`et al. 2002). These sites are involved in exon recognition
`through the binding of members of a subfamily of splicing
`
`www .rnajournal.org 1611
`
`

`

`Aartsma-Rus and van Ommen
`
`TABLE 1. Overview of exon skipping applications
`
`Target gene
`
`Protein
`
`Targef
`
`Goal
`
`Application
`
`Referenceb
`
`APOB
`
`Apolipoprotein B
`
`Bcf-X
`
`Bcl-xS and Bcl-xl
`
`COL7A1
`
`Collagen type 7
`
`DMD
`
`Dystrophin
`
`5' SS Bcl-xl exon
`
`3' SS and BP exon 27 Knockdown of
`APOB 100 isoform
`lsoform switching
`from anti- to
`pro-apototic Bcl-x
`Allele specific
`knockdown
`
`El Exon 70
`
`3' SS, 5' SS, El
`numerous DMD
`exons
`
`Reading frame
`restoration leading
`to partially functional
`dystroph ins
`
`FOLH1
`
`Prostate-specific
`membrane antigen
`
`5' SS exon 1, exon 6,
`or exon 18
`
`IL-5Ralpha
`
`ll-5 receptor-a
`
`3' SS or 5' SS exon 9
`
`MyDBB
`Tau
`
`MyD88
`Tau
`
`TNFRSFIB
`
`TN Fa 2 receptor
`
`5' SS exon 2
`5' SS or 3; SS
`exon 10
`Exon 7 and 8
`
`Ttn
`
`WT1
`
`Titin
`
`WT1
`
`5' SS exon 45, 79,
`37, 47
`IE Exon 5
`
`lsoform switching
`from transmembrane
`to cytoplasmatic form
`lsoform switching from
`transmembrane to
`soluble form
`lsoform switching
`Restore normal ratio
`3R/4R tau isoform
`lsoform switching from
`transmembrane to
`soluble form
`lsoform specific
`knockdown
`lsoform switching to
`pro-apoptotic form
`
`•ss, splice site; IE, intra-exonic; BP, branch point site.
`bAn overview of the most important publications for each application.
`cFrontotemporal dementia and parkinsonism linked to chromosome 17.
`
`Retard atherosclerosis
`
`Khoo et al. (2007)
`
`Cancer therapy
`
`Mercatante et al.
`(2001, 2002)
`
`Goto et al. (2006)
`
`Dystrophic
`epidermolysis bullosa
`therapy
`DMD therapy
`
`van Deutekom et al.
`(2001)
`Aartsma-Rus et al.
`(2003)
`Lu et al. (2003)
`Aartsma-Rus et al.
`(2004a)
`Alter et al. (2006)
`Prostate cancer therapy Williams and Kole
`(2006)
`
`Asthma therapy
`
`Karras et al.
`(2000, 2001)
`
`Anti-inflammatory
`FTDP-17c therapy
`
`Vickers et al. (2006)
`Kalbfuss et al. (2001)
`
`Rheumatoid arthritis
`therapy
`
`P. Sazani (pers. comm.)
`
`Functional analysis of
`isoforms
`leukemia therapy
`
`Seeley et al. (2007)
`
`Renshaw et al. (2004)
`
`factors, known as serine and argmme rich proteins (SR
`proteins) (Stojdl and Bell 1999). These SR proteins have
`one or several RNA domains able to bind to loosely defined
`sequence motifs that make up ESEs. SR proteins then
`recruit the essential U2AF and Ul snRNP splicing factors to
`the 3' polypyrimidine tract and 5' splice sites, respectively,
`and thus facilitate splicing. The importance of ESEs is
`underlined by the finding that intraexonic point mutations
`often result in exon skipping on the RNA level, rather than
`yielding no or missense amino acid changes as deduced
`from DNA analysis (Cartegni et al. 2002). Famous exam(cid:173)
`ples are the neurofibromatosis type 1 gene and the ataxia
`telangiectasia mutated gene, where a significant number of
`mutations lead to exon skipping (Teraoka et al. 1999; Ars
`et al. 2000; Wimmer et al. 2007). In addition, predicted
`nonsense mutations in in-frame exons of the DMD gene
`occasionally turn out to actually induce exon skipping and
`a BMD phenotype, indicative that these nonsense muta(cid:173)
`tions disrupt ESE sites (Shiga et al. 1997; Ginjaar et al. 2000;
`
`Tuffery-Giraud et al. 2004; Disset et al. 2006). As SR
`protein binding to ESEs is essential for exon inclusion,
`blocking ESEs with AONs would be expected to result in
`exon skipping. Matsuo and colleagues indeed showed that
`blocking the ESE they had identified in exon 19 resulted in
`exon skipping (Pramono et al. 1996). ESE motifs are only
`loosely defined because, even though inclusion of the exon
`in mRNA is essential, strict motifs would interfere with the
`main task of an exon, i.e., to encode protein information.
`Therefore, targeting ESEs reduces the chance of mistarget(cid:173)
`ing. Software packages, such as RESCUE-ESE, ESEfinder,
`and the PESX server, predict putative ESE sites (Fairbrother
`et al. 2002; Cartegni et al. 2003; Zhang and Chasin 2004;
`Smith et al. 2006), which facilitates the design of exon(cid:173)
`internal AONs. We have now designed almost 150 exon(cid:173)
`internaJ AONs, of which nearly 70% are effective in
`inducing the skipping of 39 different DMD exons (2,