BMC Molecular Biology
`
`BioMed Central
`
`Open Access
`Research article
`Antisense oligonucleotide induced exon skipping and the dystrophin
`gene transcript: cocktails and chemistries
`Abbie M Adams†1, Penny L Harding†1, Patrick L Iversen2,
`Catherine Coleman1, Sue Fletcher1 and Steve D Wilton*1
`
`Address: 1Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Western Australia and 2AVI BioPharma,
`4575 SW Research Way, Corvallis, Oregon, USA
`
`Email: Abbie M Adams - aadams@cyllene.uwa.edu.au; Penny L Harding - pharding@cyllene.uwa.edu.au;
`Patrick L Iversen - piversen@avibio.com; Catherine Coleman - coleman@cyllene.uwa.edu.au; Sue Fletcher - sfletch@cyllene.uwa.edu.au;
`Steve D Wilton* - swilton@cyllene.uwa.edu.au
`* Corresponding author †Equal contributors
`
`Published: 2 July 2007
`
`BMC Molecular Biology 2007, 8:57
`
`doi:10.1186/1471-2199-8-57
`
`This article is available from: http://www.biomedcentral.com/1471-2199/8/57
`
`Received: 13 February 2007
`Accepted: 2 July 2007
`
`© 2007 Adams et al; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Background: Antisense oligonucleotides (AOs) can interfere with exon recognition and intron
`removal during pre-mRNA processing, and induce excision of a targeted exon from the mature
`gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA
`processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin
`gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead
`to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting.
`Results: Although many dystrophin exons can be excised using a single AO, several exons require
`two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when
`applied individually, yet pronounced exon excision was induced in transfected cells when the AOs
`were used in select combinations, clearly indicating synergistic rather than cumulative effects on
`splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2
`different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and
`phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a
`consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations
`in exon skipping efficiency with overlapping AOs, were also seen when the corresponding
`sequences were prepared as phosphorodiamidate morpholino oligomers.
`Conclusion: The combination of 2 AOs, directed at appropriate motifs in target exons was found
`to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This
`combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to
`induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs
`composed of 2'-O-methyl modified bases, was also observed when these same sequences were
`evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established
`with one chemistry may be applied to the other.
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`Background
`Antisense oligonucleotides (AOs) can be used to modify
`gene expression through the induction of a variety of
`mechanisms. Oligodeoxyribonucleotides can be used to
`target a gene transcript for RNaseH induced degradation,
`whereas oligomers composed of modified bases can redi-
`rect gene expression through RNA silencing [1], suppress-
`ing specific mRNA translation [2-4], enhancing mRNA
`stability [5] and redirecting pre-mRNA splicing patterns
`[6].
`
`Protein-truncating mutations in the dystrophin gene typi-
`cally lead to Duchenne muscular dystrophy (DMD), the
`most common severe childhood form of muscle wasting
`(review, [7]). Although the size of this gene, with 79 exons
`spanning some 2,400 kb, and distribution of expression
`have posed major challenges for gene repair or replace-
`ment strategies, these features have opened other avenues
`for intervention, such as AO induced exon skipping. Tar-
`geted removal of selected exons can excise or by-pass pro-
`tein-truncating mutations from the dystrophin pre-mRNA
`during the splicing process. The application of AOs to
`induce targeted exon skipping in the dystrophin gene has
`been reported by several groups, examining different ani-
`mal models [8-11], regions of the human dystrophin gene
`transcript [12-14] and a variety of AO chemistries [12,15-
`18]. We have recently reported a comprehensive list of
`AOs that can induce skipping of all dystrophin exons,
`excluding the first and last exons [19]. Many exons could
`be targeted for excision from the mature dystrophin
`mRNA with a high level of efficiency and in some cases,
`two exons were consistently removed using a single AO,
`suggesting tight coordination of recognition of these
`exons with intron removal. However, some exons were
`found to be extremely difficult to dislodge, despite the
`evaluation of many AOs directed to the target exon. These
`"recalcitrant" exons could be efficiently excised from the
`mature mRNA in response to some select combinations of
`apparently ineffective AOs.
`
`AOs composed of 2'-O-methyl modified bases on a phos-
`phorothioate backbone (2OMeAO) have been used
`extensively to induce targeted exon skipping in the dys-
`trophin gene transcript and have some advantages over
`the phosphorodiamidate morpholino oligomers (PMO),
`including ease and cost of production, and efficient in
`vitro delivery when administered as cationic lipoplexes.
`However, PMOs appear to be better suited to in vivo appli-
`cation, where the increased stability and cellular uptake of
`uncomplexed compounds allows for higher levels of sus-
`tained dystrophin exon skipping, as well as an excellent
`safety profile [20-22]. In this report, we describe optimi-
`zation and excision of recalcitrant dystrophin exons from
`the mature mRNA using AO cocktails for enhanced effi-
`ciency and/or specificity. The use of either 2OMeAOs or
`
`PMOs does not seem to influence exon skipping trends,
`indicating optimization of AO design with the 2OMe
`chemistry should be directly applicable to PMOs.
`
`Results and discussion
`We have designed AOs capable of individually excising 77
`of the 79 exons from the dystrophin gene transcript [19],
`yet no universal motif has been identified as a reliable tar-
`get for the consistent redirection of dystrophin pre-mRNA
`splicing. The rationale in our approach to AO design was
`to first direct AOs at motifs obviously implicated in exon
`processing and recognition, such as the acceptor and
`donor splice sites, as well as exonic splicing enhancers as
`predicted by ESEfinder [23]. Once some dystrophin exon
`skipping was observed in AO-transfected human myo-
`genic cells, a series of overlapping AOs were then designed
`to target that area, in an attempt to develop more effective
`AOs. In many cases, a single AO was eventually developed
`that would induce substantial levels of targeted exon skip-
`ping, and the study would then move to another dys-
`trophin exon. Since this is regarded as a work in progress,
`the dystrophin exons were regarded as a reference point
`and classified into four types based upon the ease of exci-
`sion from the mature mRNA [19]. Type 1 dystrophin
`exons are removed most efficiently (greater than 30%
`after in vitro transfection at 100 nM), Type 2 are less easily
`dislodged, while Type 3 exons are poorly excised. Type 4
`dystrophin exons are "special cases", where either a single
`AO removed multiple exons, or multiple AOs are required
`to excise a single targeted exon.
`
`We observed that approximately two out of three AOs
`applied to the dystrophin pre-mRNA were able to induce
`some exon skipping, but there was considerable variation
`in levels of induced exon removal, relative to the intact
`dystrophin transcript. In some cases, the exon excision
`only occurred at low levels, or was sporadic. The fre-
`quency of these sporadic exon skipping events was greater
`than that observed in untreated cells, indicating some
`interference with the splicing process. However, the lack
`of reproducibility, or any dose-dependant responses indi-
`cated further refinement was essential.
`
`Dystrophin exon 20 skipping
`Dystrophin exon 19 was one example of a Type 1 exon
`that was very easy to dislodge from the mature mRNA,
`with every AO directed at acceptor, donor and intra-
`exonic splicing enhancers able to induce some dystrophin
`exon skipping [24,25]. In contrast, the following exon in
`the dystrophin pre-mRNA was one that proved a much
`greater challenge. Eighteen AOs were prepared to anneal
`to predicted splice motifs across exon 20 (Additional file
`1, Fig 1a), and the majority induced either no, or sporadic
`exon skipping (data not shown). Several different AO
`cocktails were then evaluated and found to induce some
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`exon 20 skipping, although consistent variation in effi-
`ciency between the different preparations was evident
`(Figures 1b–f). One preparation, consisting of equal
`amounts of H20A(+44+71) and H20A(+147+168), was
`found to be more efficient at exon 20 excision than other
`combinations and induced the shortened transcript to lev-
`els of 36% when compared to the full length product after
`transfection at 25 nM (Figure 1e). Eventually, a single AO,
`H20A(+39+69), was developed to induce exon 20 skip-
`ping, but this was still not as efficient as the cocktail of
`H20A(+44+71) and H20A(+147+168) (Figure 1g). Fur-
`ther combinations of AOs were evaluated including AO
`H20A(+39+69), the most active when used individually,
`and the non-overlapping AO from the most effective AO
`cocktail, H20A(+147+168). Unexpectedly, this particular
`combination was consistently not as efficient at exon 20
`excision and only resulted in 13% of the shortened prod-
`uct (Figure 1f), even though there is considerable overlap
`between the annealing coordinates of H20A(+44+71) and
`H20A(+39+69). These two AOs targeted essentially the
`same predicted ESE motifs, with the only differences
`being H20A(+44+71) extended only one base into a puta-
`tive SC35 motif, and H20A(+39+69) overlapped one base
`of a predicted SRp40 motif. Although it seems unlikely
`that these subtle annealing differences would contribute
`to the variation in exon excision efficiency, we have
`shown that the length of an AO is an important parameter
`in design [34]. While "longer is better" in many instances
`of single AO-induced exon skipping, this does not hold
`true in all cases. This presumably arises through the mask-
`ing of motifs involved in exon silencing or recognition, or
`influence on secondary structure of the AO. It would
`appear that combinations of the "best" AOs may not nec-
`essarily lead to the optimal AO cocktails, at least for dys-
`trophin exon 20, currently classified as a Type 4 exon.
`
`The same trend in inducing exon 20 skipping was
`observed in the mdx mouse dystrophin gene transcript.
`Individual AOs were essentially ineffective and one AO
`combination was most efficient at exon excision [26].
`However, it was of interest to note that the AO annealing
`coordinates of the AOs in the "mouse cocktail" were dif-
`ferent from those directed at the human dystrophin gene
`transcript, indicating that it may not be possible to extrap-
`olate AO design from one species to another. Further
`comparisons of induced exon skipping between the
`mouse and human dystrophin gene transcripts are cur-
`rently underway.
`
`Dystrophin exon 65 skipping
`Exon 65 was another dystrophin exon that was difficult to
`exclude from the mature mRNA. Eight AOs were designed
`to the donor and acceptor splice sites, as well as putative
`SR protein binding sites as predicted by ESEfinder [23]
`(Additional file 2 and Figure 2a). Exon 65 AOs were indi-
`
`AO induced excision of human dystrophin exon 20Figure 1
`
`AO induced excision of human dystrophin exon 20.
`Primary human myogenic cultures were transfected with cat-
`ionic lipoplexes:AO preparations at the concentrations indi-
`cated and incubated for 24 hours before total RNA was
`extracted. Nested RT-PCR was undertaken across exons
`18–26. The full-length and exon 20 deleted transcripts are
`represented by products of 1336 and 1094 bp, respectively.
`(a) Overview of AO annealing coordinates across dystrophin
`exon 20. (b) AO cocktail 20.1 and 20.2, (c) AO cocktail 20.1
`and 20.3, (d) AO cocktail 20.2 and 20.5, (e) AO cocktail 20.3
`and 20.4, (f) AO cocktail 20.5 and 20.6, (g) single AO 20.6.
`
`vidually transfected into cultured human myogenic cells
`as cationic lipoplexes at concentrations of up to 600 nM,
`but all except one AO failed to induce readily detectable
`exon 65 skipping (Figure 2b). There was a trace of the
`shortened transcript missing exon 65, less than 5%, in
`response to transfection with AO 65.3 (H65A(+26+50))
`at 600 nM, but exon 65 excision was not observed at lower
`concentrations. However, while some AO cocktails were
`found to be essentially inactive, other combinations were
`very efficient at inducing exon 65 skipping. The AO cock-
`tail of H65A(-11+14) and H65A(+26+50) was able to
`induce 65% skipping after in vitro transfection at 100 nM
`(data not shown). Subsequent titrations studies with this
`AO cocktail indicated 35% exon 65 exclusion after in vitro
`transfection at total concentrations of 10 nM, with 20%
`skipping at 5 nm and 8% skipping at 2 nM, that is 1 nM
`of each AO. Approximately 2% exon excision was detected
`after 0.5 nM transfection, the lowest concentration tested
`(Figure 2c). Individually, these AOs were unable to induce
`any exon 65 skipping after transfection at concentrations
`hundreds of fold greater, again indicating some synergy
`between these compounds. We originally reported a 3 AO
`cocktail for exon 65 removal [19], but subsequent studies
`indicated equivalent efficiency with the combination of
`only H65A(-11+14) and H65A(+26+50).
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`missing the targeted exon as well as 17+18, 34+35 and
`54+55 respectively [19].
`
`The induced skipping of exon 10 was distinct from these
`cases in that larger blocks of exons were involved, and the
`resultant patterns were somewhat variable. However,
`upon combining H10A(-06+15) with H10A(+98+119) or
`H10A(+130+149), specific exon 10 excision could be
`achieved, although there was still some evidence of addi-
`tional shortened transcripts induced by the individual
`AOs.
`
`Trends in AO cocktail design
`Additional file 4 provides an overview of the predicted
`ESE splice motifs masked by the AOs reported in this
`study, with an indication of the maximum score and
`number of motifs shown in brackets. One feature that was
`common to all effective AO cocktails directed to exon 10,
`20 and 65 was the targeting of predicted SC35 motifs by
`both AOs in the mixture. We do not propose that pre-
`dicted SC35 motifs are the most important targets for
`induced exon skipping, as 8 out of 41 AOs targeting Type
`1 exons, that are removed at high efficiency, do not appear
`to be directed at any predicted SC35 motifs [19]. The rel-
`evance of the SC35 motifs to induced exon skipping is not
`known and requires further investigation.
`
`Dystrophin exon 67 had previously been classified as a
`Type 3 exon, that is, only low levels of exon skipping were
`induced by the single AO, H67A(+22+47) [19]. This AO
`was predicted to anneal to 3 SC35 motifs and, while sub-
`stantial exon skipping was induced after transfection at
`600 nM, weaker skipping at 300 nM and there was no
`detectable skipping at lower concentrations. However,
`upon combination of H67A(+22+47) with either of two
`other AOs directed at the acceptor, H67A(-10+17), or
`donor site H67D(+11-14), 50% exon 67 skipping was
`generated after transfection at 50 nM, with 42% and 23%
`exon 67 skipping induced after transfection at 10 and 2
`nM, respectively (data not shown). The AOs directed at
`the exon 67 acceptor and donor sites were shown to be
`inactive when used individually at concentrations of 600
`nM, and neither was directed to predicted SC35 motifs.
`Although the exon 67 cocktails do not conform to the
`observation that AOs targeting SC35 motifs are more
`effective in cocktails, this may reflect on the AO common
`to both cocktails, H67A(+22+47), which targeted three
`SC35 motifs and exhibited substantial exon skipping
`potential when applied at high concentrations.
`
`AO cocktails that induced the most pronounced exon
`skipping did not necessarily block donor and acceptor
`sites, and other splicing motifs, nor did the most effective
`AO combination correlate with the total number of ESE
`sites targeted. For example, the most effective cocktail for
`
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`AO induced excision of human dystrophin exon 65Figure 2
`
`AO induced excision of human dystrophin exon 65.
`Primary human myogenic cultures were transfected with cat-
`ionic lipoplexes:AO preparations at the concentrations indi-
`cated and incubated for 24 hours before total RNA was
`extracted. Nested RT-PCR was undertaken using primers
`directed to exons 63–68. The full-length and exon 65 deleted
`transcripts are represented by products of 618 and 416 bp,
`respectively. (a) Overview of AO annealing coordinates
`across dystrophin exon 65, (b) Single AO transfection with
`65.5, 65.3 and 65.7, (c) AO cocktail 65.1 and 65.3. Note that
`the upper concentration of AO:lipoplex administered to
`these cells was 10 nM in total.
`
`Dystrophin exon 10 skipping
`The use of AO cocktails is not limited to enhancing exon
`removal from the dystrophin mRNA. In the case of
`human dystrophin exon 10, a combination of 2 AOs was
`required for specific exon excision. As with other recalci-
`trant exons, several AOs described in Additional file 3,
`were unable to induce any detectable removal of exon 10
`when applied individually, except for H10A(-05+16),
`which only excised exon 10 together with blocks of flank-
`ing exons (data not shown). Several dystrophin tran-
`scripts missing exons 10–12, 9–12, 9–14 and 9–15 were
`sporadically detected, with 9–12 and 9–14 being most
`commonly observed. These dystrophin transcripts are in-
`frame and, extrapolating from a mildly affected Becker
`muscular dystrophy patient missing exon 9–22 [27],
`would be expected to produce a shorter dystrophin that
`could be of near-normal function. Two AOs, H10A(-
`09+16) and H10A(-05+24) that overlapped H10A(-
`05+16) had no effect on the processing of the dystrophin
`transcript (data not shown).
`
`Some AOs directed at other parts of the dystrophin gene
`transcript had been shown to remove one or two exons in
`addition to the target, and this was assumed to reflect
`highly coordinated processing of both exons. Targeting
`human or canine dystrophin exon 8 always leads to tran-
`scripts missing exons 8 and 9 [11,19], whereas directing
`AOs to human exons 17, 34 or 54 induces transcripts
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`exon 65, H65A(-11+14) and H65A(+26+50) masked
`three SC55 motifs, a single SF2/ASF and a SRp40 site. In
`contrast, a less effective AO combination directed at the
`same exon was directed at 4 SC35 sites, 2 SF2/ASF, 2
`SRp40 and a single SRp55 motif. Exons 10 and 65 are
`removed by AOs directed at the acceptor site and internal
`ESE's, while exon 20 AOs anneal to internal ESE's.
`H10A(+98+119) and H65A(+63+87), annealed to all four
`predicted SR binding sites (SF2/ASF, SC35, SRp40 and
`SRp55), and these AOs were inactive when used individu-
`ally.
`
`AO chemistry comparisons in cocktails
`The 2OMeAOs have some advantages over other AO
`chemistries, including PMOs, in that they can be readily
`synthesized in-house and may be efficiently transfected
`into cultured myogenic cells as cationic lipoplexes. PMOs
`are not readily taken up by cultured cells, unless high
`transfection concentrations are applied, scrape loading is
`employed [28,29], or the PMOs are coupled to cell pene-
`trating peptides to enhance delivery [30-32]. We have
`undertaken other comparisons between the 2OMeAOs
`and the PMOs directed at the mdx mouse nonsense muta-
`tion in exon 23 and observed that the PMOs offer much
`greater potential in vivo [16,17,33]. We now extend these
`studies to other targets, including dystrophin exons that
`had been difficult to displace and have found the same
`trends in exon skipping are observed with both chemis-
`tries.
`
`PMOs directed to the same coordinates as the 2OMeAOs,
`H20A(+44+71) and H20A(+147+168), were unable to
`induce any detectable skipping of human exon 20, despite
`being transfected individually at concentrations of up to
`20 µM (Figure 3a and 3b). However, as with the corre-
`sponding 2OMeAOs, a combination of the two PMOs
`resulted in substantial exon 20 skipping after transfection
`at a total concentration of 5 µM, that is 2.5 µM of each
`PMO (Figure 3c). These particular PMOs did not carry a
`peptide tag to enhance delivery, hence the transfection
`concentration was substantially higher than that used by
`the 2OMeAO cationic lipoplexes. Subsequent experi-
`ments have shown that exon 20 could be excised with the
`PMO cocktail at total concentrations as low as 1 µM (data
`not shown), whereas the individual PMOs could not
`induce skipping at concentrations 40-fold higher.
`
`Chemistry comparisons in AO design
`We have previously reported that the length of an AO can
`play an important role in the ability of that compound to
`induce exon skipping [34]. One mdx mouse model of
`muscular dystrophy has a nonsense mutation in exon 23
`[35], and has been useful in optimizing AO design
`[8,9,34,36] and comparing different chemistries [15,17].
`We have shown that the donor splice site of mouse dys-
`
`Induced skipping of human dystrophin exon 20 with PMOsFigure 3
`
`Induced skipping of human dystrophin exon 20 with
`PMOs. Primary human myogenic cultures were transfected
`with PMOs at the concentrations indicated and incubated for
`24 hours before total RNA was extracted. Nested RT-PCR
`amplified between exons 18 and 26. The full-length and exon
`20 deleted transcripts are represented by products of 1336
`and 1094 bp, respectively. (a) Individual PMOs directed to
`exon 20. (b) PMO cocktail directed to exon 20. Note that
`the total concentration of PMOs is shown, ie, 5 µM indicates
`2.5 µM of each PMO.
`
`trophin exon 23 was an amenable target for redirecting
`splicing and demonstrated a 25 mer, M23D(-7+18) was
`more efficient than a shorter AO, M23D(+2-18). The latter
`compound was in turn found to be more efficient at
`inducing exon 23 excision than 2 longer AOs, M23D(+12-
`18 and M23D(+7-23) [34]. This same hierarchy of exon
`23 skipping was also observed when these sequences were
`prepared as PMOs, coupled with cell penetrating peptides,
`and evaluated in cultured cells. RNA was extracted 24
`hours after transfection, and as can be seen in Figure 4, the
`25 mer, M23D(+7-18), induced superior exon skipping to
`the 20 mer, which in turn was more effective than both 30
`mers, M23D(+12-18) and M23D(+7-23). At later time-
`points (data not shown), it was possible to establish that
`PMO M23D(+7-23) was marginally more effective at exon
`23 removal than the other 30 mer, confirming the trends
`shown by the 2OMeAOs [34]. In addition, the shortest
`products in Figure 4 correspond to dystrophin gene tran-
`scripts missing exons 22 and 23, products that are also
`generated in response to transfection with 2OMeAOs and
`previously reported [9,36]. Thus trends seen with the
`2OMeAOs in terms of ranking of efficiency of overlapping
`AOs, synergy when applied in cocktails and the more
`effective compounds also promoting exclusion of exons
`22 and 23, correlate to those observed with the PMOs.
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`Induced skipping of mouse dystrophin exon 23 with overlap-ping PMOs directed at the donor splice siteFigure 4
`
`
`Induced skipping of mouse dystrophin exon 23 with
`overlapping PMOs directed at the donor splice site.
`Immortalised H2K-mdx myotubes were transfected with
`PMOs at the concentrations indicated and incubated for 24
`hours before total RNA was extracted. Nested RT-PCR was
`undertaken to amplify exons 20 and 26. The full-length and
`exon 23 deleted transcripts are represented by products of
`901 and 688 bp respectively. The band of 542 bp corre-
`sponds to dystrophin gene transcripts missing both exons 22
`and 23 and has been previously reported [9, 36].
`
`All sequences developed as 2OMeAOs and shown to
`induce skipping of Type 1 exons [19] have now been pre-
`pared as PMOs (n = 41). These PMOs were shown to
`induce the same dystrophin exon removal patterns as
`those generated by the corresponding 2OMeAOs after
`transfection in either primary human myogenic cultures
`or muscle explants (data not shown). Detailed compari-
`sons of sub-optimal PMOs to other dystrophin targets has
`not been undertaken, as the perfect concordance observed
`to date between the exon skipping trends with 2OMeAOs
`and PMOs would suggest that this would be unnecessary
`and a waste of resources. Our efforts are being directed to
`fine-tuning the next series of PMOs likely to enter clinical
`trials, as well as improving upon the efficiency of excision
`of Type 2 and 3 dystrophin exons, through improved AO
`design, and/or the use of AO cocktails. It will also be of
`interest to revisit some of the Type 1 dystrophin exons that
`are efficiently removed with a single AO, to ascertain if the
`application of AO cocktails can further enhance exon
`removal at very low AO concentrations. Although the use
`of AO cocktails will require the synthesis of two different
`compounds, these AOs would be used as a single prepara-
`tion and safety evaluation would be undertaken on the
`combination. Should an AO cocktail be ten-fold more
`effective that an optimal single AO to induce targeted
`exon excision, there would be clear production and cost
`benefits. Perhaps more importantly, the use of AO cock-
`tails may address safety and efficacy issues in that lower
`amounts of an AO preparation will need to be adminis-
`tered.
`
`Conclusion
`AO induced exclusion of dystrophin exons during pre-
`mRNA processing offers a potential treatment for remov-
`ing or by-passing protein-truncating mutations that lead
`to DMD. For induced exon skipping to be a viable ther-
`
`apy, the most effective AO preparations must be devel-
`oped so that minimal amounts can be administered.
`Many dystrophin exons could be efficiently removed from
`the mature mRNA by the intervention of a single AO dur-
`ing dystrophin pre-mRNA processing. Some exons require
`the action of two AOs, often ineffective when used indi-
`vidually, which somehow act in a synergistic fashion, pre-
`sumably through prevention of spliceosome assembly by
`altering pre-mRNA folding or masking crucial protein
`binding sites. The SC35 motif appears to play some role as
`an amenable target for AO cocktails, but this association
`is not absolute. Clearly, there are many parameters
`involved in interfering with the pre-mRNA splicing proc-
`ess remaining to be elucidated. The trends in splice inter-
`vention may be seen with AOs composed of 2 different
`chemistries, 2OMeAO and PMO.
`
`Methods
`AO design and synthesis
`2OMeAOs were prepared on an Expedite 8909 Nucleic
`acid synthesiser using the 1 µMol thioate synthesis proto-
`col. AOs were designed to anneal to splicing motifs at the
`intron: exon boundaries, as well as ESE motifs predicted
`by the web based application, ESEfinder[23].
`
`PMOs, and PMOs conjugated to the cell penetrating pep-
`tide, were synthesized by AVI Biopharma (Corvallis, Or).
`AO nomenclature is based upon that described by Mann
`et al, 2002 [36]. The first letter designates the species, the
`number indicates the exon, the second letter specifies
`Acceptor or Donor splice sites, with the -/+ and numbers
`representing the annealing coordinates in the intronic and
`exonic domains respectively. For example, H65A(-11+14)
`would anneal across the acceptor site of human dys-
`trophin exon 65, specifically to the last 11 bases of intron
`64 and the first 14 nucleotides of exon 65.
`
`Culture and transfection – Primary human myoblasts
`The preparation of primary human myoblasts is described
`by Rando and colleagues, 1994 [37]. Primary human
`myotubes were transfected in Opti-MEM (Invitrogen), 48
`hrs after seeding, with Lipofectamine 2000 (L2K): AO at
`1:1 w:w ratio according to manufacturer's instructions
`(Invitrogen). For each experiment, transfections were
`repeated three times to confirm reproducibility.
`
`Culture and transfection – H-2Kb-tsA58 (H2K) mdx
`myoblasts
`H2K-Mdx myoblasts [38] were cultured as described by
`Mann and colleagues 2001 [9]. AOs were transfected with
`Lipofectin:AO at 2:1 w:w ratio, 24 hrs after seeding. Lipo-
`fectin was used according to manufacturer's instructions
`(Invitrogen, Melbourne). All transfections occurred in
`duplicate wells and were repeated three times to ensure
`consistency.
`
`Page 6 of 8
`(page number not for citation purposes)
`
`

`

`BMC Molecular Biology 2007, 8:57
`
`http://www.biomedcentral.com/1471-2199/8/57
`
`Acknowledgements
`The authors received funding from the National Institutes of Health
`(RO1NSO44146-02), the Muscular Dystrophy Association USA
`(MDA3718), the National Health and Medical Research Council of Australia
`(303216), Parent Project (UK) and the Medical and Health Research Infra-
`structure Fund (Western Australia).
`
`Molecular analysis
`RNA extraction and RT-PCR have been described previ-
`ously [8,9]. Briefly, RNA was purified from duplicate cul-
`tures using an acid phenol extraction, before a one step
`RT-PCR was undertaken using specific primers, template
`and the Invitrogen Superscript III. After 30–35 cycles of
`amplification, an aliquot was removed and subjected to
`nested PCR using inner primer sets. Details of all primers
`used in these experiments are available upon request. The
`identity of the RT-PCR products was confirmed by direct
`DNA sequencing [39]. Estimates of relative exon skipping
`efficiency were performed using the Vilber Lourmat
`Chemi-Smart 3000 system with Chemi-Capt software for
`image acquisition and Bio-1D software for image analysis.
`
`Authors' contributions
`AMA and PLH carried out the AO cocktail design and cell
`transfections, participated in the data acquisition and
`image preparation. PLI assisted in PMO design and sup-
`plied test compounds. CC participated in the AO cocktail
`evaluation. SF and SDW conceived the study, participated
`in its design and drafted the manuscript. All authors read
`and approved the final manuscript.
`
`Additional material
`
`Additional file 1
`Sequences of AOs designed and evaluated for inducing excision of
`human dystrophin exon 20. Reference numbers may be used to orientate
`the annealing coordinates shown in Figure 1.
`Click here for file
`[http://www.biomedcentral.com/content/supplementary/1471-
`2199-8-57-S1.xls]
`
`Additional file 2
`Sequences of AOs designed and evaluated for inducing excision of
`human dystrophin exon 65. Reference numbers may be used to orientate
`the annealing coordinates shown in Figure 2.
`Click here for file
`[http://www.biomedcentral.com/content/supplementary/1471-
`2199-8-57-S2.xls]
`
`Additional file 3
`Sequences of AOs designed and evaluated for inducing excision of human
`dystrophin exons 10 and 67.
`Click here for file
`[http://www.biomedcentral.com/content/supplementary/1471-
`2199-8-57-S3.xls]
`
`Additional file 4
`Summary of AO combinations evaluated for targeted removal of
`human dystrophin exons. Serine-arginine rich protein (SR) binding
`scores for each AO in the combination are shown. The efficiency of exon
`removal is indicated (*). (n/a-not applicable).
`Click here for file
`[http://www.biomedcentral.com/content/supplementary/1471-
`2199-8-57-S4.xls]
`
`2.
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`3.
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