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`I
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`Neurology India (!M)
`v. 56 · n~ . 3 (Jul-Sept 2008)
`Genera, Collection
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`ISSN - 0028-3886
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`Official publication of Neurological Society of India
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`Neurology India
`
`Print ISSN· 0028-3886, E-ISSN. 1998-4022
`The Official journal of the Neurological Society of India
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Review Article
`
`Exon skipping and Duchenne muscular dystrophy: Hope,
`hype and how feasible?
`
`Steve D. Wilton, Susan Fletcher
`Centre for Neuromuscular and Neurological Disorders, Molecular Genetic Therapy Group, University of Western Australia, Australia
`
`Duchenne muscular dystrophy (DMD), the most common
`and serious form of childhood muscle wasting is generally
`caused by protein-truncating mutations in the large DMD
`gene. Specific removal of an exon from a defective DMD
`gene transcript has the potential to allow synthesis of a
`semi-functional dystrophin, thereby reducing the severity and
`presumably progression of muscle wasting. The efficacy of
`this treatment will vary greatly between the different mutations
`that preclude the synthesis of a functional dystrophin .
`Restoration of the reading frame from a large multi-exon
`genomic deletion, typically greater than 36 exons, may lead
`to synthesis of a protein with only partial function and limited
`clinical benefit, whereas excising a nonsense mutation in a
`redundant exon should generate a near normal dystrophin.
`A clinical trial has recently confirmed proof-of-principle
`that exclusion of Exon 51 from human dystrophin mRNAs,
`carrying frame-shifting deletions adjacent to this exon, results
`in dystrophin expression. No major side-effects after local
`administration of the antisense oligomer were reported.
`Additional trials are underway, targeting the same exon
`but using an oligomer of different backbone chemistry. If
`functional dystrophin synthesis is demonstrated, and safety
`issues are addressed, subsequent trials will involve systemic '
`delive_ry .. Great chall~nges ~re ahead, some technical; ]
`establishing an effective delivery regimen, some ethical; I
`choosing subsequent targets for therapy, and others of an
`administrative and regulatory nature.
`
`Key words: Becker muscular dystrophy, clinical trials,
`Duchenne muscular dystrophy, exon skipping, morpholino
`oligomer, personalized genetic medicines
`
`.....
`
`~ac_kgroun_~
`
`Duchenne and Becker muscular dystrophy (DMD and
`1MD) arc allelic muscle wasting conditions arising
`'.>m mutations in the large DMD gene al Xp21.2.1 11 The
`
`most common, serious and progressive form, DMD,
`is caused by inactivation of the DMD gene product.
`Affected individuals appear normal al birth and clinical
`symptoms may be observed between the ages of two
`lo three years. Approximately soc,Vci of DMD males do
`nol walk until after the age of 18 months, and exhibit
`signs of retarded motor development, including a
`waddling gait, difficulty running and jumping and calf
`enlargement. Muscle wasting is relentlessly progressive
`in a symmetrical fashion, with joint contracturcs
`an important clinical sign. As the process of muscle
`regeneration becomes overwhelmed, regenerating fibers
`arc less frequent and the replacement with adipose and
`connective tissue contributes to pseudo-hypertrophy
`of some muscles. Affected individuals are typically
`non-ambulant by the age of 12 years, and in some
`cases as early as seven years (revicw,12-~ 1). Contracturcs
`develop as the disease progresses, and in the absence
`of optimal care, including corticosteroid treatment,
`physical therapy and nocturnal assisted venlilation,Pil
`most patients succumb lo the disease by the age of
`20 years as the result of respiratory and/or cardiac
`complications.
`In contrast, BMD patients present wilh a spectrum of
`SDVDrity, from liorderline DMD to asymptomatic.lfi, 71 It
`has lieen estimated that BMD occurs at only 1()% the
`incidc!nce or DMD, most likely through the inability
`to rncognize and diagnose particularly mild cases
`prior.111 Although many BMD individuals present with
`some symptoms bctwecm the ages of fivn to 15 years,
`by definition a IlMIJ patic!nt will rn111ai11 ambulanl until
`age 1Cl years or longer. Some patients dnspite a delc!tion
`within the dystrophin coding sequonc<!, present with
`no evidence of pathology, or nlnvaled sorum crnatine
`kinase, a sensitive marker of muscle damage.I'll
`The reading frame rule, proposed liy Monaco
`cl al., lends to hold trne for Llw majority of DMD
`and 13MD cases.l<J,WI Duclwnno muscular dystrophy-
`
`Professor Steve Wilton
`Molecular Genetic Therapy, University of Western Australia, Centre for Neuromuscular and Neurological Disorders, Australian Neuromuscular Research Institute
`'
`4" Floor "A" Block, QE II Medical Centre, Ned/ands 6009, Western Australia . E-mail: swilton@meddent.uwa.edu.au
`
`254
`
`Neurology India I July-September 2008 I Vol 56 I Issue 3
`
`Th is mate-rial w;as co,pded
`at t he NLJMand m ay b e
`S;ubj ect US Copyright Law s
`
`

`

`Wilton, et al.: Exon skipping and DMD
`
`causing mutations, either frame-shifting deletions
`or duplications, nonsense mutations or splice motif
`errors lead lo the loss of a functional DMD gene
`product. Becker muscular dyslrophy typically results
`from in-frame deletions in lhe DMD gene lhal allow
`synthesis of an internally lruncalocl, lrnl functional
`
`prolein. 1'1•1111 The variation in severily reflects lhe
`extent and location of the deletion. Large mulli-exon
`deletions or the loss of crucial functional domains
`typically lead to the more severe phenotype, whereas
`loss of in-frame exons within Lhe cenlral rod domain
`appears lo have lillle or no consequences.1 11 ·151 The
`general rule is lhal lhe loss of 3G or more exons is
`associated with a severe phenotype, regardless of lhe
`reading frame, suggesting a minimum size for functional
`dyslrophin.l thl The lack of dyslrophin disrupts the
`link between the aclin cyloskelelon and basal lamina,
`compromising lhe sarcolemma (review,1 171). Dyslrophic
`muscle fibers are prone lo injury during force genera lion
`and repealed damage loads lo muscle loss and
`subsequent fibrosis.
`However, rare dyslrophin-positive fibers accumulate
`in muscles of many DMD patients and in animal
`models of muscular dystrophy.1rn•2111 This remarkable
`phenomenon is the result of naturally occurring
`allernalive dyslrophin lranscripls lhal bypass the
`DMD gene lesion. Although Lhe precise mechanism is
`unknown, lhe loss of multiple exons from the mRNA,
`flanking the DMD genomic deletion, has the polenlial
`lo restore the reading frame of the dystrophin transcript
`in the reverlanl fibcrsY1-2:q In the mdx mouse, an animal
`model of muscular dystrophy, this mechanism generated
`in-frame DMD gene transcripts missing multiple exons,
`typically 20 or more, including Exon 23 that contains
`the dyslrophin inactivating nonsense mulalion.12~1
`Tho skeletal muscle-specific dyslrophin isoform plays
`a crucial role, stabilizing the sarcolemmal171 through tho
`functional domains, the primary actin-binding silos al
`the amino terminal encoded by Exons 2, 4 and 5, and
`weak aclin binding between oxons 35 and 44, and tho
`cysleine rich ~-dystroglycan binding site encoded by
`Exons G2-G9Y 51 The central rod domain appears lo be
`variably dispensable, as demonstrated by the mutations
`in mildly aff cctod or asymptomatic BMD patients,liiJ
`and tho existence of naturally occurring rovorlanl
`fibers. Hence, a logical slralegy lo reduce the severity of
`DMD caused by dyslrophin-lruncaling mutations is lo
`manipulate primary gene transcript processing so as lo
`produce an in-frame mRNA capable of being translated
`into a functional BMD-likc prolein.1221
`
`Dystrophin: An Ideal Candidate for
`Transcript Manipulation
`
`Manipulation of dyslrophin pre-mRNA processing
`might appear a formidable challenge, considering the
`
`Neurology India I July-September 2008 I Vol 56 I Issue 3
`
`size and complexity of expression of this gene. The
`strategy demands lhal one or two oxons be excluded
`from a 2.4Mb DMD gene transcript during the
`simultaneous expression and processing of thousands
`of other gene lranscripls. However, many DMD gene
`features that have proved a challenge lo cell and gene
`replacement or repair therapies for DMD arc regarded
`as positive attributes for lranscripl manipulation,
`termed 'cxon skipping'.
`Tho DMD gene is the largest known and consists
`of 79 exons spanning some 2.4 Mbp,l 2
`GI much
`loo large lo be incorporated into viral vectors
`currently available for clinical applications. The
`wild type proloin-coding region is in excess of 11
`Kb, incompatible with the capacity of most viral
`vectors. Tho identification of a very mildly affected
`BMD patient with an in-frame deletion of almost
`half the gone prompted tho construction of a vector
`containing the dyslrophin "minigeno" ,127I Elegant
`studies have shown that multiple dyslrophin
`domains can be trimmed lo create microdystrophin
`-:1 111 Those constructs arc compatible
`isoforms.12 11
`wilh current viral vectors, lending impetus to gene
`replacement studies and confirming lhal there
`arc conditionally redundant domains within tho
`dyslrophin protein,t:l 1I Detailed studios have shown
`that restoration of micro-dystrophin expression al
`tho sarcolemma docs nol necessarily correlate with
`functional rescueY21
`Under the control of multiple promoters, tho
`predominant dyslrophin transcript is expressed in
`skeletal muscle as a malure 14 kb mRNA.l 1-25•:ni It has
`been eslimalcd that lG h elapse during the processing
`of a single dyslrophin pre-mRNA, during which lime
`79 cxons must be spliced from lhc primary gene
`transcript,1 141 Possibly because of this major effort in
`gene processing, tho mature DMD gone transcript is
`present at very low levels and has been estimated to
`only constitute 0.01-0.001 % of the total mRNAY51
`The full length transcript encodes a protein with four
`major domains and the reading frame is represonlod
`in cartoon form in Figure la. Tho most common type
`of dyslrophin mutation is a deletion of one or more
`exons, which may disrupt the reading frame with
`catastrophic consequcncos.1 10.:wJ However, nonsense
`mutations where a single base change alters a codon
`into a premature protoin-lerminalion signal [Figure
`lb], micro-insertions/deletions to disrupl the reading
`frame [Figure le] or nucleotide changes that disrupl
`prc-mRNA processing so that an cxon is lost, or
`inlronic sequences arc retained in the mature mRNA
`[Figure ld] have been reported. A BMD-like gone
`message is depicted in Figure le, where the removal
`of the "END" nonsense mu talion, or the "ND" frame(cid:173)
`shift bypasses the gene lesion, and permits the resl
`of the gene message lo bo translated.
`
`255
`
`Th is mat erial w as copcied
`at the NLM and maybe
`~ubject USCo pcyright Laws
`
`

`

`]
`
`...
`
`Wilton, et al.: Exon skipping and DMD
`
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`
`Figure 1: Schematic representation of normal and defective dystrophin
`transcripts. Crucial functional domains, actin binding and the Cys(cid:173)
`rich ~·dystroglycan binding domains are represented by gray and
`black boxes respectively. DMD-causing mutations are indicated by
`underlining: (b) nonsense, (c) frame-shift deletion and (d) frame-shift
`insertion. An in-frame deletion, typical of BMD (underlined), and the goal
`of targeted exon skipping is shown (e)
`
`Targeted Exon Skipping
`
`Some 15 years ago, Kole and colleagues directed
`anlisens e oligomers (AOs) with 2'-O-melhyl modified
`bases on a phosphorolhioale backbone (2OMeAOs) lo
`cryptic splice silos that arose from inlronic mutations
`in the ~-globin geneY71 Upon masking the cryptic splice
`silo, Lhe splicing machinery defaulted to recognition of
`Lhe normal 13-globin splice silns. Induced clyslrophin
`exon skipping is similar in principle, except that
`normal splice motifs are largolod to mask tho oxon
`from the splicing machinery, lnading lo nxc;lusion of
`the exon from the mRNJ\. In this 111a11111:r, a 11011s1!11se
`mutation can be rnmov1:d or lhn rnading f'ramn can lw
`restored around d1!lolio11s or insnrlions in 1111! DMD
`gene transcript. Although 1:xo11 skippi11g is tlw most
`common consequenco of splic11 motif mulalio11s, so11w
`splice d efects lead lo llw activation of 011n or mor1:
`cryptic s plice sites.
`In Lh e golden rntrievor model of muscular dystrophy
`(GRMD),f:w1 Lhe majority of dyslrophin transcripts arn
`out-of-frame, sin ce the inlron G acceptor splice sile
`mutation result s in loss of Exon 7 from Lhe mature
`mRNA.J :rnr However, sensitive and specific PCR
`conditions detected near normal length dystrophin
`transcripts containing all bul Lhe first five bases of
`Exon 7 as some transcript s arose from the recognition
`of the first AG in Exon 7 .1-io1 Had this Lranscripl been
`in-frame , it is possible th a t the severity of the disease
`may have been mitigated somewhat in these dogs .
`Human dyslrophin splice site mutations and activation
`of cryptic splice sites have been reported, including one
`in intron 26 which appears lo have arisen inclopendenlly
`at least twice (www.dmd.nl).1-ii1
`
`256
`
`Th is materia I w as c.o,pied
`at t he N LM a.nd may be
`Soubject US Copy right Law s
`
`Early studies
`A 2'-O-me lhyl oligoribonucleolide was designed
`lo largel an internal motif in Exon 19 in a cell-free
`sys lem ,14 21 followed by skipping of dys lrophin Exon
`19 using an oligodeoxynucleolide in lymphoblasloid
`cells.1 431 Although the donor and acceptor sites were not
`targeted , these oligomers induced Exon 19 skipping .
`These oligomers mirnicked Lhe eff ecl of the Kobe DMD
`mutation, where an intra-exonic del e tion removed
`key splice recognition motifs and led lo the omission
`of Exon 19 from the gene transcript.lH,451 Analysis of
`Lhe Leiden Muscular Dystrophy Pages (www.dmd.nl)
`revealed that removal of one of only 12 exons should
`restore the reading frame in 75% of dyslrophin exon
`deletion patienls.1401 This group reported resloralion of
`the reading frame in a number of different normal and
`DMD patient cell lines, and mosl importantly, showed
`the generation of dyslrophin protein by Western blotting
`and immunofluoresccnt sludies.l47·51J
`We commenced dyslrophin exon skipping studies
`using Lhe mdx mouse,1521 an animal model carry ing a
`nonsense mulalion in dystrophin Exon 23. 15:11 Although
`Lhe mdx mouse is commonly referred Lo as a model of
`DMD, this is nol strictly accurate, since these animals
`do not present the severe muscle wasting seen in
`DMD boys. Despite this limitation, the mdx mouse
`offers an excellent molecular model of a dystrophin
`mu talion, and through detailed histological studies, il is
`apparent that the diaphragm develops severe dyslrophic
`pathology as the animal ages.1 541
`We Lransfcc ted three 2OMeAO cationic lipoplexes
`inlo primary mdx myogenic cells.1551 The scrambled
`AOs and Lh c oligomer directed al Lhe acceptor splice
`site had no delectable effect on dyslrophin pre-mRNA
`processing. However, the 20mer directed to the donor
`silo was able to induce Exon 23 skipping in a dose(cid:173)
`clr!pendenl manner. A series of overlapping AOs were
`subsequenlly designed lo both donor and acceptor sites
`and, whil1! llw acceptor remained an unresponsive
`largel, Lho officiency of exon skipping was improved
`by s1:l!:cling a 20mer targeted to the donor site_l5'WI The
`us11 of a block, co-polymor F127 resulted in functional
`amounts of dyslrophin uxprnssion in Lhu libialis
`anl nrior after intramuscular clolivory of llw same AO.1"'1f
`Ollwr formulations havu also lw11n shown lo onlwnco
`2OMeAO deliveryY,' 1 "1.1;21
`DNA oligonwrs arn of limited polonlial for induced
`exon skipping, since this cl10111islry induces [{Nase
`H ac tivity, leading lo mRNA degrndalion. Perhaps
`more signifi cantly, oligonwrs of lhis c lwmislry will
`be rapidly degraded by nuclemms .W11 Although 2OMe
`AOs have proved most effective, since !hoy are
`more nuclease resistant and do nol support RNaseH
`activity, several other oligonwr chomislries have
`bee n evaluated, including peptide nucleic acids
`(PNAs),fGHiq locked nucleic acids (LNAs),1" 1•071 2'-
`
`Neurology India I July-September 2008 I Vol 56 I Issue 3
`
`

`

`Wilton, et al.: Exon skipping and DMD
`
`1
`•
`;
`
`O-(2-Melhoxy)elh y l-moclifiecl (MOE)l 1; 01 and
`phosphorodiamidale morpholino oligomers (PMOs).
`1i;n.i;!iJ The PNAs were nol inilially rcporlcd lo be
`effcctive,1"4
`111 however, a rcccnl rcporl showed some
`exon skipping. 1G
`1>1 The LNAs induced robusl exon
`skipping, bul lhe exceedingly high Tm indicalcd great
`polcnlial for off-largcl effccls due lo cross-annealing lo
`rclalcd scqucnces,l 1
`HI This group reporled lhat PMOs
`were nol effective and lhal 2OMcAOs were the preferred
`chemistry for clinical application.
`
`effective systemic dystrophin resloralion.17111 Numerous
`peptides were screened in an elegant transgenic mouse
`model and the most promising candidate was then
`evaluated for delivery of the PMO targeted lo dyslrophin
`Exon 23 in the mdx mouse. There is no evidence of
`immune responses lo the peptides to dale (unpublished
`data), but further investigation into nonspecific or toxic
`eff ccls is ongoing.
`
`Target Site Selection and Oligomer Design
`
`Oligomer Delivery
`
`Al though in vilro uptake of 2OMeAO calionic lipoplexes
`into myogenic cells is efficient, these compounds have
`poor uptake in the absence of delivery reagents . To
`facilitate a comparison between 2OMcAOs and PMOs,
`we annealed a sense strand leash to the PMO, thereby
`allowing the duplex lo be complexed with a cationic
`liposome, before lransfection into cultured cclls.w11 IL
`became apparent thal while uncomplexcd PMOs were
`not taken up efficiently in vitro, PMO:leash:cationic
`lipoplcxes induced robust and sustained exon skipping.
`The PMO uptake by cultured cells after applicalion al
`high concenlralions and scrape loading to facilitate
`uptake has been rcportcd.1 701
`We reported that a single intramuscular injection of
`a PMO in saline was able lo induce strong dyslrophin
`expression, six weeks after administration.1 1;111 The
`compound, injected into 11-day-old mice before the
`peak of muscle degeneration al 18-21 days,1 711 was able
`lo prevent muscle breakdown, as evidenced by normal
`muscle morphology and a statistically significant
`reduction in fibers with central nucleation, a marker
`of muscle regeneration. Subsequent reports showed
`that systemic delivery of the PMO into the mdx
`mouse restored dystrophin expression in skeletal and
`smoolh muscle, although the heart was refractory to
`lrcatment.l1in.721
`A major improvement in the efficacy of exon skipping
`after systemic delivery was achieved through the use
`of J>MOs coupled to cell-penetrating peptidcs,17=1
`741
`The ability of PMO-pcptidc conjugates to induce exon
`skipping has been demonstrated in vitro, including
`human and mouse muscle explants,1 751 GR.MD canine
`model of muscular dystrophy17 61 and the mdx 4cv1 771
`mouse model (Milrpanl, unpublished data). Body(cid:173)
`wide dystrophin expression was restored in mdx mice
`by intraperitoneal administration of the PMO-cell(cid:173)
`penetraling peptide P007 conjugate as four, once-weekly
`doses of 5 mg/kg.1n1 Although exon skipping was not
`demonstrated in cardiac muscle, the reduction in
`skeletal muscle damage lowered the scrum creatinc
`kinase to near-normal levels. Recent development of a
`peptide that mediates PMO-induccd cxon skipping in
`cardiac muscle has removed the remaining barrier lo
`
`,
`
`Neurology India I July-September 2008 I Vol 561 Issue 3
`
`Although in vitro studies evaluating human dyslrophin
`exon skipping and work in animal models have proved
`promising, there are four major challenges lo exon
`skipping therapy for DMD that must be overcome.
`Achieving effective system delivery has been an obstacle
`lo many nucleic acid therapies, as is selection of an
`oligomcr chemistry that can safely induce sustained
`re-direction of dyslrophin expression after long-term
`administration. In addition, it will be necessary to
`design many different oligomers to restore the reading
`frame around mutations , including non-deletion
`mutations, across the DMD gene, and undertake safely
`and toxicology studies.
`The oligomers designed to excise selected dystrophin
`exons in three animal models of muscular dystrophy,
`mdx and mdx 4cv mice and the GRMD dog, have not
`provided us with any obvious parameters for optimal
`targeting. A donor splice site was most amenable when
`targeting Exon 23 for removal from the mdx mouse
`DMD gene transcript, while canine dystrophin Exons
`G and 8 were excised by oligomers directed to ESEs
`and the acceptor site respectively. Mouse dyslrophin
`Exons 52 and 53 could only be efficiently removed
`using a combinalion of two and three AOs respectively
`(unpublished data).
`Every dystrophin cxon, excluding the first and last,
`can be excised from the mature human DMD gene
`lranscript. Aartsma Rus and colleaguesl 47
`l reported on
`114 oligomers that were evaluated for the excision of
`35 dystrophin exons. We reported a preliminary draft
`of AO sequences capable of removing each human
`dystrophin exon and found that a substantial proportion
`of compounds could induce some exon skipping, albeit
`lo a variable degrcc.1rn1 Some AOs could induce readily
`detectable exon skipping after in vitro lransfcclion as
`lipoplcxes at concentrations of 10 nM, while others
`induced sporadic or very low levels of exon skipping
`after administration at 600 nM. Clearly, the most
`applicable compounds for the clinic will be those that
`are effective at the lowest concentrations, with more
`easily achievable therapeutic thresholds and lower risk
`of off-target eff ecls.
`We, and others have shown that an oligomcr
`may induce targeted exon skipping, even when the
`compound includes mismalches.1n4,no1 We demonstrated
`
`257
`
`This materia l \vascopie<l
`3tth,e NLM a n<! m 3y b.e
`:.ubj ect US Copyright L3WS
`
`

`

`Wilton, et al.: Exon skipping and DMD
`
`skipping of Exon 19 from th e human and mouse
`dystrophin lranscripls using mismatched AOs, lml only
`al high concentrations. Clearly, cross-reaction lo related
`sequences is a possibility and il will be mosl important
`lo design effective compounds Lhal excise lhe largol
`exon at low concentrations. We reported lhal displacing
`an oligomer by a few nucleotides dramatically alters
`exon skipping activily.1110-11 11
`Consequently, we have devoted considerable effort
`to design oligomers capable of inducing robust exon
`skipping at low concentrations, and it has become
`appare nt that targe ting obvious splice motifs will
`not guarantee induced exon skipping.'7'11 Despite
`early success in excluding Exon 23 from lhe mouse
`dystrophin mRNA by targeting the donor splice
`sile,'""."7 1 directing an oligomer lo the identical
`coordinates in the human DMD gene transcript had no
`effect on dyslrophin processing.11111 Indeed, the human
`donor splice sites are not generally preferred targets,
`with only lwo exons out of the 77 having donor splice
`sites as the optimal target motif17u1 and lhe majority of
`exons were efficiently removed by oligomers directed lo
`ESEs, intra exonic motifs that enhance exon recognition.
`A small number of exons that proved difficult lo remove
`showed satisfactory levels of exon skipping when
`targeted by combinations of oligomers.1112·1u1 The effect of
`AO cocktails is clearly synergistic, as particular AOs that
`are ineffective individually can induce exon skipping
`when applied al very low concentralions.1'111
`The AO length emerged as playing an important
`role in oligo design. In many cases, increasing the
`length from 25 lo 30 nucleotides conferred suhslanlial
`increases in exon skipping nfficinncy, far oulwnighing
`lhe additional cost synllll!sis. 11111 Increasing OIII! oligomnr
`from 25 lo 30 n11cleolid1:s incrnased nxon skipping
`efficiency fourfold,J1 11 1 howc!ver, oligonll!r 11:nglh must
`be determined on illl exon-by-1:xon basis. Inc:rnasing
`lhe length of the oligonwr targeting llw n1011s1:
`dyslrophin Exon 2:1 donor sit<: from 2!> lo :IO hasns was
`counterproductive. Our data suggests that Lill! optimal
`oligomcr length for exon skipping is lwtwc!nn 2:1 and
`30 nucleotides, rcgmdlnss of whether tlw 2OMn/\O or
`•11"1
`PMO chemistry is usecl.1 111
`
`Exon Skipping: Clinical Implementation
`
`In the first reported clinical trial of exon skipping
`for DMD, a DNA oligomer targe ted lo Exon 19 was
`administered intravenously lo a single DMD patient
`missing dyslrophin Exon 20. 11141 Despite showing
`appropriate dyslrophin exon skipping in lymphocytes,
`induced Exon 19 skipping and restoration of dyslrophin
`synthesis in muscle appeared equivocal. Several
`parameters could have conlriliuled lo this, including
`the nature of the oligomer chemistry, dosage, liming
`and duration of the regimen.
`
`More recently, a Phase Ia trial nol only addressed
`the primary safely concerns but also conclusively
`clemonstralecl restoration of dyslrophin expression in
`the most common subset of DMD deletion mutation
`palienls.11151 Intramuscular injection of a 2OMeAO
`(PRO051) targeted lo Exon 51 was carried out in
`four DMD individuals with different deletions in Lhe
`major mutation hotspot. Exci s ion of Exon 51 restored
`the reading frame in all participants, and dyslrophin
`expression was unequivocally demonstrated al the site
`of injection by immunofluornscence, Western blolling
`and RNA studies. A Phase I trial of a PMO targeting
`Exon 5111w1 is ongoing, and as yet, no data is available.
`This protocol differs from that by van Deulekom
`cl aJ.,1 1101 in terms of chemistry, oligomer sequence and
`largot muscle.
`
`Non-deletion Mutations
`
`Exon skipping development has focused on restoring
`the reading frame for the most common type of
`dyslrophin mutation: genomic deletions. Exon 51 was
`selected as the target for the first clinical trial , because
`removal of this exon would benefit a large percentage of
`DMD patients. However, exon skipping can lie applied
`lo other types of dyslrophin mutations, and perhaps it
`would be more accurate lo consider exon skipping as
`a generic platform of intervention for DMD. Although
`genomic deletions of one or more exons are the most
`common DMD-causing mutations, cases arising from
`exonic duplications, nonsense mutations, splice
`motif detects, micro-insertions or deletions may be
`more amenable to exon skipping. Aartsma-Rus and
`colleaguesl 1171 recently restored the reading frame in
`DMD patients with genomic duplications. If one copy
`of a single-exon duplication could be removed, the
`rnsultanl dyslrophin transcript should he translated
`inlo a normal protein. Although apparently simplistic,
`this may prove a technical challenge. It would appear
`that larg!!ling a duplicated exon generally rnsulls in the
`removal of both exons from the lranscripl, leading to
`disrnplion of tlw n:ading franHl unless additional exons
`arn rnmoved (Wilton, 11np11l>lishncl olisnrvatio11s).1'171
`Ceneralion of nor111al transcripts from ollwr ()MIJ
`dyslrophin gmws by solnclc!d nxon nxcl11sio11 is possihlc!.
`i'snudo-exon incorporntion in tho mature: ()Ml) ge1w
`transcript arisns from inlronic liascl changns which
`activate a set of cryptic splico motifs that nwd tlw
`criteria for nxon recog11ilio11. The 1:xc1lssivn intronic
`component of the IJMD gmw may conlrihule lo the
`incidence of pseudo-exons as a cause: of DMD, rnporled
`lo occur al a frequency of up to 4'¾1.1'1111 IJclspile tlw rarity
`of pseudo-exon inclusion, such mu lat ions arn perhaps
`ideal candidates for exon skipping, since 1!xcl11sion
`would result in a normal clyslrophin mRNA.1'1111
`We