By-passing the nonsense mutation in the 4CV mouse model of muscular dystrophy by induced exon skipping

Abstract

Background

Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd^mdx–4Cv/J (4^CV) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.

Methods

A series of 2′-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.

Results

Compared to the original mdx mouse studies, induction of exon skipping from the 4^CV dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4^CV myogenic cells and in vivo, after a single intramuscular injection.

Conclusions

By-passing the protein-truncating mutation in the 4^CV mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo. Copyright © 2008 John Wiley & Sons, Ltd.

Introduction

Duchenne muscular dystrophy (DMD), a devastating neuromuscular disorder characterized by progressive muscle wasting, and proximal muscle weakness, arises from the absence of functional dystrophin. Dystrophin links the actin cytoskeleton to the extracellular matrix via a complex of proteins embedded in the sarcolemma and plays a pivotal role during muscle contraction 1–3. Loss of dystrophin renders muscle fibres vulnerable to membrane damage during contraction. Progressive loss of muscle fibres, with inflammatory cell infiltration and fibrosis eventually overwhelms the regenerative capacity of the muscle.

The human dystrophin gene is the largest known, and the major muscle isoform consists of 79 exons, spanning 2.4 million bp. Most mutations in DMD patients are intragenic deletions or duplications, accounting for approximately 60% and 8% of all DMD patients, respectively 4–7. Point mutations, including nonsense and splice motif mutations, as well as small insertions/deletions that disrupt the reading frame, are responsible for 25–35% of all cases 7, 8. DMD mutations typically disrupt the reading frame, thereby preventing synthesis of functional dystrophin. Becker muscular dystrophy (BMD) is also caused by mutations in the dystrophin gene but this milder allelic condition is generally caused by gene defects that do not disrupt the reading frame and allow production of shorter, but partially functional, protein. Depending upon the position and nature of the mutation, some cases of BMD may only be diagnosed late in life, and present with very mild or no symptoms 9, 10, whereas others may present as borderline DMD and lose ambulation around the age of 15 years 11, 12.

AO-induced exon skipping studies initially targeted different splice motifs of exon 23 in the muscular dystrophy mouse model (mdx), with the aim of restoring protein expression 13, 14. The defect in the mdx mouse is a naturally occurring nonsense mutation in dystrophin exon 23. Despite limitations, including a mild clinical phenotype, the mdx mouse has been widely used in developing potential therapies for DMD, including exon skipping 15–22, gene and cell replacement 23, 24, and premature translation termination suppression 25.

In the present study, the B6Ros.Cg-Dmd^mdx–4Cv/J (4^CV) muscular dystrophy mouse 26, carrying a nonsense mutation in exon 53 of the dystrophin gene, was used to evaluate AO-induced dual exon skipping in a region of the dystrophin gene within the major human dystrophin deletion hot-spot. By-passing the 4^CV mutation, and maintaining the reading frame, requires removal of both exons 52 and 53 from the mature dystrophin gene transcript. A series of AOs were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. AOs of two different chemistries, 2′-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs), and phosphorodiamidate morpholino oligomers (PMOs) conjugated to a cell-penetrating peptide (P007) 27, 28 were compared.

Materials and methods

AOs and primers

AO nomenclature is based on that described by Mann et al.18. Sequences and composition of AO treatments are described in Table 1. 2OMeAOs were synthesized on an Expedite 8909 Nucleic Acid synthesizer (Applied Biosystems, Foster City, CA, USA) using the 1 µmol thioate synthesis protocol. AOs were designed to anneal to either exonic sequences or exon/intron junctions of mouse dystrophin exons 52 or 53. PMOs conjugated to an arginine-rich, cell penetrating peptide (P007) 27, 28 were synthesized by AVI BioPharma (Corvallis, OR, USA). Primers for RT-PCR and sequencing analysis were synthesized by Geneworks (Adelaide, Australia) and are listed in the Supporting information (Table S1).

Table 1. Sequences, annealing coordinates, length and GC content of all AOs targeting exons 52 and 53

AO	Length (bp)	Annealing coordinates	Exon number	Sequences	GC content (bp)	%
1	25	M52A + 17 + 41	52	5′-UCC AAU UGG GGG CGU CUC UGU UCC A-3′	14	56
2	30	M52A − 15 + 15	52	5′-AUC UUG CAG UGU UGC CUG AAA GAA AAA AAA-3′	10	33
3	30	M52D + 15 − 15	52	5′-CAU UAA GAG ACU UAC UUC GAU CAG UAA UGA-3′	10	33
4	30	M52A + 22 + 51	52	5′-AAU GAG UUC UUC CAA UUG GGG GCG UCU CUG-3′	15	50
5	30	M52A + 32 + 61	52	5′-GGG CAG CAG UAA UGA GUU CUU CCA AUU GGG-3′	15	50
6	30	M52A + 42 + 71	52	5′-UUC AAA UUC UGG GCA GCA GUA AUG AGU UCU-3′	12	40
7	31	M53A + 39 + 69	53	5′-CAU UCA ACU GUU GUC UCC UGU UCU GCA GCU G-3′	15	48
8	30	M53A − 15 + 15	53	5′-UCU GAA UUC UUU CAA CUG GAA UAA AAA UAA-3′	7	23
9	30	M53D + 15 − 15	53	5′-AUG CUU GAC ACU AAC CUU GGU UUC UGU GAU-3′	12	40
10	30	M53A + 29 + 58	53	5′-UGU CUC CUG UUC UGC AGC UGU UCU UGA ACC-3′	15	50
11	30	M53A + 49 + 78	53	5′-UUA ACA UUU CAU UCA ACU GUU GUC UCC UGU-3′	10	33
12	30	M53A + 59 + 88	53	5′-GUU GAA UCC UUU AAC AUU UCA UUC AAC UGU-3′	9	30
13	30	M53A + 69 + 98	53	5′-CAG CCA UUG UGU UGA AUC CUU UAA CAU UUC-3′	11	37
14	30	M53A + 19 + 48	53	5′-UCU GCA GCU GUU CUU GAA CCU CAU CCC ACU-3′	15	50
15	30	M53A − 25 + 5	53	5′-UUC AAC UGG AAU AAA AAU AAG AAU AAA GAA-3′	6	20
16	30	M53A + 129 + 158	53	5′-CCA UGA GUC AAG CUU GCC UCU GAC CUG UCC-3′	17	57
17	30	M53A + 151 + 180	53	5′-CUA CUG UGU GAG GAC CUU CUU UCC AUG AGU-3′	14	47
18	30	M53A + 176 + 205	53	5′-UCU GUG AUC UUC UUU UGG AUU GCA UCU ACU-3′	11	37
19	30	M53D + 5 − 25	53	5′-UUU UAA AGA UAU GCU UGA CAC UAA CCU UGG-3′	10	33
20	25	M53A − 25 − 1	53	5′-CUG GAA UAA AAA UAA GAA UAA AGA A-3′	5	20
21	25	M53A − 20 + 5	53	5′-UUC AAC UGG AAU AAA AAU AAG AAU A-3′	5	20
22	25	M53A + 69 + 93	53	5′-AUU GUG UUG AAU CCU UUA ACA UUU C-3′	7	28
23	25	M53A + 74 + 98	53	5′-CAG CCA UUG UGU UGA AUC CUU UAA C-3′	10	40
24	25	M53A + 99 + 123	53	5′-CCU GUU CGG CUU CUU CCU UAG CUU C-3′	13	52

Animals

4^CV (B6Ros.Cg-Dmd^mdx–4Cv/J) congenic mice, obtained from the Jackson Laboratory (Bar Harbor, ME, USA), were raised and supplied by the Animal Resources Centre, Murdoch, Western Australia. Animal housing and transport followed guidelines from National Health and Medical Research Council (Australia). The use of animals was approved by the Animal Ethics Committee of University of Western Australia (approval number 03/100/572).

Cell culture and AO transfection

Immortalized mdxH2K^b-tsA58 mouse cells (H-2K mdx) were propagated and transfected as described previously 29. Primary myoblast cultures were prepared from 2–4-day-old 4^CV pups and the procedure was adapted from Rando et al.30. Limb muscles from four pups were dissected, homogenized, and incubated at 37 °C for 30 min with dissociating enzyme mix containing 2.4 units/ml dispase (Invitrogen, Victoria, Australia), 5 mg/ml collagenase Type II (Invitrogen), and 2.4 mM CaCl₂ in Dulbecco's modified Eagle's medium (Invitrogen). After centrifugation, the cell pellet was added to a 75-cm² tissue culture flask with 10 ml of proliferative media and incubated for 1 h. Non-adherent cells were removed and seeded into 75-cm² tissue culture flasks coated with 100 µg/ml matrigel. When nearly confluent, cells were seeded into 24-well plates coated with 50 µg/ml poly D-lysine solution and matrigel, and incubated for 48 h before transfection. Duplicate wells were transfected with 2OMeAO lipoplexes using Lipofectin (Invitrogen) at a ratio of 2 : 1 Lipofectin to AO. Briefly, Lipofectin was mixed with OptiMEM (Invitrogen) to a final volume of 200 µl and incubated for 30 min at room temperature. The 2OMeAO, which had been diluted to 200 µl in OptiMEM, was then combined with Lipofectin : OptiMEM and the mixture incubated for a further 30 min, before addition of OptiMEM to a final volume of 1 ml and subsequent addition of 500 µl aliquots to each well. Transfected cells were incubated for 48 h before RNA was extracted for analysis.

Intramuscular administration

Oligomers, in physiological saline, were injected into tibialis anterior (TA) muscles at doses indicated. Each experiment included at least one saline only injection as a negative control. The animals were anaesthetized and sacrificed by cervical dislocation at indicated time points after the injection, and muscles were removed and snap-frozen in pre-cooled isopentane, before being sectioned and prepared for RNA and protein studies.

RNA extraction, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and DNA sequencing

RNA was harvested from H-2K mdx cultures, 4^CV cultures and frozen sections tissue blocks using Trizol (Invitrogen), according to the manufacturer's protocol. One-step RT-PCR was undertaken using 120 ng of total RNA as template, in a 12.5 µl reaction for 30 cycles of amplification. After the reverse transcription step for 30 min at 55 °C, the reaction was heated to 94 °C for 2 min before the primary thermal cycling rounds of 94 °C for 40 s, 60 °C for 1 min, and 68 °C for 1 min. Nested PCR was then carried out on 1 µl of the primary amplification reaction using AmpliTaQ Gold (Applied Biosystems, Foster City, CA, USA). Cycling conditions for the secondary PCR were 94 °C for 6 min to activate the polymerase, followed by 20 cycles of 94 °C for 40 s, 60 °C for 1 min, and 72 °C for 1 min. PCR products were separated on 2% agarose gels in TAE buffer and the images captured on a CHEMISMART-3000 (Vilber Lourmat, Marne-La-Vallee, France) gel documentation system. Bands of interest were re-amplified directly from the agarose gel 31 and the sequencing templates were purified using UltraClean spin columns (Mobio Laboratories, Carlsbad, CA, USA) and then sequenced on an ABI 377 automated sequencer using BigDye v3.1 terminator chemistry (Applied Biosystems).

Western blot analysis

Protein extracts were prepared as weight per volume of treatment buffer containing 125 mM Tris/HCl, pH 6.8, 15% sodium dodecyl sulphate, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM dithiothreitol, bromphenol blue, and protease inhibitor cocktail (Sigma, St Louis, MO, USA) to cell pellet or mouse muscle cryostat sections. Samples were then vortexed and sonicated briefly, heated at 95 °C for 5 min, before electophoretic fractionation on a 4–10% sodium dodecyl sulphate gradient gel at pH 8.8 with 4% stacking gel, pH 6.8. Densitometry of myosin bands after coomassie blue staining was undertaken to facilitate equivalent protein loading. Extracts from AO treated cultures or muscle cryosections from treated animals (2.75 mg) and control muscle (0.275 mg) were loaded onto a second polyacrylamide gel electrophoresis gel for western blotting. Proteins were transferred from the gel to nitrocellulose membranes (Amersham Biosciences, Castle Hill, Australia) overnight at 18 °C, at 290 mA. Dystrophin was visualised using NCL-DYS2 monoclonal anti-dystrophin antibody (Novocastra, Newcastle-upon-Tyne, UK) as described previously 21. Images were captured on a Vilber Lourmat CHEMISMART-3000 gel documentation system. The percentage of dystrophin restoration was calculated according to dystrophin expression in control cells after normalization for myosin loading.

Tissue preparation and immunofluorescence

TA muscles were taken from mice and snap-frozen in isopentane, pre-cooled in liquid nitrogen. Dystrophin was detected in 6 µm unfixed cryostat sections using NCL-DYS2, an antibody that reacts strongly with C-terminus of dystrophin. Immunofluorescence was performed using the Zenon Alexa Fluor 488 labelling kit (Invitrogen), according to the manufacturer's protocol, except for the initial fixation step. The primary antibody was diluted at 1 : 10 and sections were counterstained with Hoechst (Sigma) at the dilution of 1 : 10 000 to visualize nuclei. Sections were viewed under Olympus IX 70 inverted microscope (Olympus, Tokyo, Japan) and images were captured on an Olympus DP70 digital camera.

Results

Single exon targeting

A panel of AOs was designed to anneal to obvious splicing motifs, including the acceptor and donor splice sites, and potential exonic splicing enhancers (ESEs) of both exons 52 and 53. Although designed to predict human ESEs, the web-based ESE prediction program, ESEfinder 3.0 32, 33, was used to identify putative ESEs in mouse dystrophin exons 52 and 53. Figure 1 shows the predicted ESEs of exons 52 and 53 and the relative annealing positions of the AOs (sequences are provided in Table 1) designed to induce exon removal. Six AOs, designed to excise dystrophin exon 52, were evaluated initially in H-2K^b-tsA58 mdx (H-2K mdx) immortalised myogenic cells 34. The use of these immortalized cells reduced animal usage and, because the 4^CV mutation in exon 53 did not alter dystrophin splicing patterns, it was assumed that design in one mouse strain should be valid for another. Subsequent experiments validated this approach. According to ESEfinder, the nonsense mutation in dystrophin exon 53 occurs in two predicted ESE motifs that are only just above the threshold: an SF2/ASF motif (score = 2.187/threshold = 1.956) and an SRp40 motif (score = 3.042/threshold = 2.67).

Figure 1. Diagrammatic presentation of predicted ESEs 32, 33; (a) mouse exons 52 and (b) exon 53. Relative annealing coordinates of oligomers are indicated

None of the AOs targeting the acceptor or donor sites of exon 52 consistently induced removal of the target (Figure 2a), whereas the AOs annealing between the coordinates + 17 + 51 efficiently excluded exon 52 from the mature dystrophin mRNA (Figure 2b). Subsequent titration studies comparing AOs 1 and 4 indicated that the latter compound was more efficient at inducing exon 52 removal at lower transfection concentrations, although there was little exon 52 skipping induced at 25 nM (data not shown). Consequently, AO 4 was selected for further studies.

Figure 2. Induced single exon skipping in H-2K mdx cells; (a) Transcripts resulting after transfection with AO 2 (typical of that obtained after transfection with AOs 3, 5 and 6); (b) Transcripts resulting after transfection with AO 4 and AO 1; (c) Transcripts resulting after transfection with AO 8 (typical of that obtained after transfection with AOs 9, 14, 15, 16 and 19–24); (d) Transcripts resulting after transfection with AO 7 (typical of that obtained after transfection with AOs 7 and 10–13); (e) Transcripts resulting after transfection with AO 17; (f) Transcripts resulting after transfection with cocktail AOs 8, 9 and 13

Excision of exon 53 proved to be a greater challenge, and although numerous AOs were designed and evaluated, no single AO tested efficiently excluded exon 53 from the mouse dystrophin mRNA. Although many AOs failed to dislodge exon 53, as shown in Figure 2c, others removed exon 53 in addition to both exons 53 and 54 (Figure 2d). Two AOs activated a cryptic splice site and resulted in partial exon 53 loss (Figure 2e). Thirteen different combinations of non-overlapping AOs were then assessed and one AO cocktail was developed to consistently induce specific exon 53 skipping (Figure 2f).

Exon 52 and 53 skipping: 2OMeAOs

As the relative efficiencies of excision of the target exons differed, it was necessary to evaluate combinations of the optimized AOs directed to exons 52 and 53 (Table 2). Four 2OMeAOs (4, 8, 9 and 13) were combined at ratios indicated (A1, A2, A3, A4 and A5) and used to transfect 4^CV cultured primary myoblasts (Figures 3a to 3c). The identity of the transcript missing exons 52 and 53 was confirmed by DNA sequencing (Figure 3d). At 2 days after transfection, the A2 and A3 cocktails appeared marginally more effective at excising both exons 52 and 53, as shown by the absence of the full-length product (Figures 3a to 3c); hence, the A2 cocktail was selected for further studies.

Figure 3. Induced exon skipping in 4^CV cells after transfection with 2OMeAO cocktails (a) A1, (b) A2 and (c) A3; (d) DNA sequencing chromatogram confirming precise splicing of exons 51–54. Transcripts resulting after transfection with (e) B1, (f, h) B2 and (g, h) B3 in 4^CV cultures; (i) western blot analysis from 4^CV cultures treated with B2 and B3 PMO cocktails

Table 2. Composition of AO cocktail: in vitro studies (2OMeAO and PMO) and in vivo studies (PMO)

Cocktail	A1	A2	A3	A4	A5
2OMe AO	1 : 3	1.66 : 1	3.33 : 1	6.66 : 1	12.33 : 1
4	150	375	460	520	555
8	150	75	46	26	15
9	150	75	46	26	15
13	150	75	46	26	15
Total concentration (mM)	600	600	600	600	600
Cocktail	B1	B2	B3	B4	B5
P007-PMO	1 : 3	1.66 : 1	3.33 : 1	6.66 : 1	12.33 : 1
4	2.5	6.25	7.7	8.8	9.25
8	2.5	1.25	0.77	0.44	0.25
9	2.5	1.25	0.77	0.44	0.25
13	2.5	1.25	0.77	0.44	0.25
Total concentration (µM)	10	10	10	10	10
Cocktail	C1	C2	C3	C4	C5
P007-PMO	1 : 3	1.66 : 1	3.33 : 1	6.66 : 1	12.33 : 1
4	10	25	30	35	37
8	10	5	3.3	1.8	1
9	10	5	3.3	1.8	1
13	10	5	3.3	1.8	1
Total amount (µg)	40	40	40	40	40

Although RT-PCR studies indicated substantial exons 52 and 53 skipping at days 3 and 5, only a trace of shortened transcript could be detected at days 8 and 9 after treatment (data not shown). No detectable dystrophin protein was observed in treated cultures at any time point. RNA and protein were analysed at day 14 after two transfections at days 0 and 9, and no exon skipping or restored dystrophin protein were observed (data not shown).

A total of 100 µg of the 2OMeAO A2 cocktail, was complexed with F127 and administered to 4^CV mice through intramuscular or intraperitoneal routes. Animals were sacrificed 3, 5 and 7 days after injection and RNA from the diaphragm and TA muscle was assessed for exon skipping. Although sporadic exon 52 and 53 skipping was detected in the TA muscle, no substantial dystrophin protein restoration could be demonstrated by either immunofluorescence or western blot analysis (data not shown).

Induced exon 52 and 53 skipping with phosphorodiamidate morpholino oligomers

We had previously found that, although 2OMeAOs were well suited for AO design and short-term in vitro RNA studies, restoration of dystrophin protein in 2OMeAO treated cultures was more problematic 35. AOs 4, 8, 9 and 13 were prepared as PMOs, coupled to the cell penetrating peptide P007. These oligomers did not induce any exon skipping when applied individually (data not shown). The combination was evaluated in 4^CV myoblast cultures and, as with the 2OMe AOs, different ratios of PMOs were assessed. The PMO cocktails B2, B3 and B4 induced the transcript missing both target exons at concentrations as low as 0.5 µM in vitro. The B2 and B3 PMO combinations comparable to the A2 and A3 2OMe combinations appeared to be the most effective mixtures, and were selected for further study (Figures 3e to 3g). Both the B2 and B3 cocktails were able to induce pronounced exon 52 and 53 skipping, 2 weeks after in vitro transfection at a concentration of 40 µM (Figure 3h), consistent with the appearance of induced dystrophin, as determined by western blotting (Figure 3i). Normalization of dystrophin according to myosin densitrometry indicates the B2 and B3 cocktails induced 11% and 8% of normal levels of dystrophin, respectively (Figure 3i; see also Supporting information, Table S2).

Evaluation off-target effects

To confirm specificity of the multi-oligo cocktail on dystrophin expression, RT-PCR was undertaken across the dystrophin gene transcript using five sets of nested primer pairs (see Supporting information) covering exons 13–70 region (Figures 4a to 4e). Figures 4a to 4e represent amplified segments of the dystrophin transcript. Although sporadic ‘revertant’ transcripts were detected in PMO cocktail treated cultures, the numbers of alternatively spliced transcripts were not greatly different from those in six untreated cultures (Figure 5).

Figure 4. RT-PCR analysis of dystrophin transcripts in untreated and PMO treated 4^CV cultures across (a) exons 13–26, (b) exons 37–50, (c) exons 58–70, (d) exons 20–35 and (e) exons 48–58

Figure 5. RT-PCR analysis of dystrophin transcripts in untreated and PMO treated 4^CV cultures across exons 20–35 indicating alternatively spliced dystrophin transcripts

In vivo studies

A single injection of 40 µg of each of the PMO cocktails, at ratios shown in Table 2, was made into the TAs of 4^CV mice. Two weeks after injection, RT-PCR on RNA extracted from injected 4^CV muscle demonstrated exon 52 and 53 skipping in all cocktail-treated samples (Figure 6a) with western blotting indicating dystrophin expression to be approximately 5–7%, based upon normalization of loading (see Supporting information, Table S2). Consistent with the RNA studies, dystrophin immunofluorescence on sections from the TAs of 4^CV mice treated with PMO cocktails C1 and C2 also showed dystrophin-positive fibres (Figures 7a and 7b).

Figure 6. (a) RNA studies; and (b) western blot analysis of muscle extracts from the 4^CV mice injected (IM) with PMO cocktails (C1 to C5)

Figure 7. Dystrophin immunofluorescence on muscle cryosections from 4^CV mice treated with cocktails (a) C1, (b) C2, (c) C3, (d) C4, (e) C5 and (f) saline; (g) untreated C57

Discussion

Oligomer design, evaluation of different chemistries and systemic delivery protocols have been extensively studied in the mdx mouse model of muscular dystrophy 14, 18–21, 36, 37. The present study describes the application of AO-induced dual exon skipping to address the primary gene lesion in another mouse model, the 4^CV mouse, whose nonsense mutation should be by-passed with the excision of exons 52 and 53. This model may be regarded as of greater relevance to the human condition because the targeted exon skipping is induced within the major hotspot for dystrophin gene deletions 38.

Acceptor and donor splice sites were initially considered obvious targets for AO-induced exon skipping, and consistent exon 23 removal was achieved with the first oligomer targeting the donor splice site 14. However, a comprehensive study targeting all human dystrophin exons indicated donor splice sites are rarely the optimal or preferred targets, and the majority of preferred targets appear to be intra-exonic motifs 39. Indeed, it appears that if a pre-mRNA site is not amenable to oligomer intervention, the application of a panel of oligomers to microwalk across that motif, or altering AO length or chemistry, is unlikely to achieve acceptable (or detectable) levels of exon exclusion 40. Similar trends were observed for the 4^CV mouse dystrophin exons 52 and 53, where individual oligomers annealing to either the donor or acceptor splices sites failed to induce any targeted exon skipping. Two overlapping oligomers, AOs 1 and 4, targeting intra-exonic motifs within exon 52, were identified that induced substantial skipping and titration studies indicated AO 4 was slightly more efficient at lower transfection concentrations.

Mouse dystrophin exon 53 proved to be much more challenging to dislodge from the mature mRNA. Eighteen oligomers were designed to target acceptor and donor splice sites, as well as intra-exonic motifs predicted by ESEfinder 32, 33. Although this program is based upon human sequences, the strong homology between mouse and human dystrophin sequences was considered sufficient justification to use this program as a starting point in AO design. Initial AO design was undertaken in an immortalized mdx mouse myogenic cell line, while awaiting establishment of the 4^CV mice colony. The sequences of dystrophin exon 52 were identical in both strains, and similar identified splice patterns were generated with AO 4 in both cell lines. Similarly, AOs that led to exon 53 skipping in the mdx cell line exhibited identical patterns in cultured cells from the 4^CV mouse. Because no aberrant splicing was observed involving exon 53 in untreated 4^CV cultures, it was presumed that the mutation did not influence splicing. This is consistent with the mutation occurring in two putative ESEs that were just above the threshold for SF2/ASF and SRp40. Once the 4^CV colony was established, all subsequent experiments were undertaken in these cells.

The majority of compounds targeting exon 53, when used individually were either ineffective, activated a cryptic splice site (a loss of 78 bases from the end of exon 53) or led to the production of dystrophin gene transcripts missing exons 53 and 54, in addition to exon 53 alone (Figures 2d to 2f). Interestingly, AO 7, which anneals to exactly the same coordinates identified as an optimal target to dislodge human dystrophin exon 53, induced mouse dystrophin transcripts missing exons 53 and 54, in addition to exon 53 excision. Several other AOs overlapping AO 7 also induced this pattern of exon removal, implying that this area may be involved in coordinated processing of both exons during pre-mRNA splicing, at least in mice. Targeting exon 53 for excision from the human dystrophin gene transcript only resulted in specific target removal, highlighting some limitations in extrapolation of oligomer design between homologous genes of different species 39. The loss of an exon flanking that targeted for excision has been reported previously. The AO targeting the mdx mouse exon 23 for removal also induced dystrophin gene transcripts missing exons 22 and 23, an out-of-frame transcript that cannot lead to dystrophin production 36. Similarly, transcripts missing exons 53 and 54 are out-of-frame, even when exon 52 was omitted and were thus considered undesirable.

Because we had previously observed that some combinations of apparently inactive AOs were able to induce highly efficient exon skipping 41, various AO cocktails targeting exon 53 were evaluated. A combination of AOs 8 and 9, targeting the acceptor and donor sites, respectively, was able to induce some exon 53 skipping, although not at a consistent and satisfactory level. Subsequently, AOs 8 and 9 were combined with other AOs (7, 10, 11, 12, 13, 14 and 16) to identify a cocktail capable of inducing specific exon 53 skipping. When AO 13 was combined with AOs 8 and 9, efficient and specific exon 53 removal could be induced after transfection at low total AO concentrations. The mixture of AOs 8 and 13 generated the same pattern induced by AO 13 alone, indicating no advantage, whereas the combination of AOs 9 and 13 only induced inconsistent exon 53 excision. This three-AO combination was necessary for both efficiency and specificity of exon skipping.

Because exon 53 was removed from the mature mRNA at a higher efficiency than exon 52, attempts were made to balance the ratio of AO 4, targeting exon 52, with AOs 8, 9 and 13, to maximize induction of in-frame transcripts. When equimolar amounts of all four oligomers were applied, the full-length transcript was observed, in addition to dystrophin mRNAs missing exon 53 and exons 52 and 53. Altering the composition of the cocktails with increased proportions of AO 4 resulted in decreased amounts of full-length product and a higher proportion of in-frame transcripts missing both exons 52 and 53. Transcripts missing only exon 52, another out-of-frame mRNA were also observed.

Although a substantial amount of exon 52 and 53 deleted transcripts were detected at days 3 and 5 after transfection with 2OMeAOs, no detectable amount of dystrophin was observed by western blot analysis (data not shown). Only trace amounts of exon skipping could be observed 8–14 days after transfection, and this also did not generate sufficient dystrophin detectable by western blotting (data not shown). It was assumed that this limitation arises from the uptake and turn-over of the 2OMeAOs in cells. The 2′-O-modified oligomers on phosphorothioate backbone increase the oligomers resistance to nuclease degradation but, unlike PMOs, which are not metabolized, 2OMeAOs are gradually broken down by nucleases 42. This was most apparent in a time-course of 2OMeAO-induced exon skipping in cultured cells, where there was substantial exon skipping 24 h after transfection but this declined substantially over the next 9 days 43. By contrast, substantial PMO-induced exon skipping transcript had been detected with less intermediate product at 14 days after transfection, in vitro44. By-passing the dystrophin gene lesion and inducing dystrophin detectable by western blotting was very inconsistent in canine cultured cells from the golden retriever muscular dystrophy model of muscular dystrophy and human dystrophic muscle explants 35, 44. However, upon the application of PMOs of the same sequences, efficient exon skipping could be induced and maintained in vitro or ex vivo to allow sufficient dystrophin synthesis to be readily detected by western blotting at 7 days after transfection 35.

The application of any oligomer to a cell has the potential to cross react with other sequences and/or exert nonspecific effects. The use of AO cocktails would potentially increase this risk and, although a detailed examination of other gene has not been undertaken, we showed that the cocktail only induced targeted changes in the dystrophin mRNA. We had noticed in other studies that the addition of oligomers appears to marginally increase the appearance of alternatively spliced transcripts 45. However, the application of four oligomers did not greatly alter the incidence of the alternative transcript in treated cells compared to untreated cultures.

The application of PMOs, coupled to an arginine rich cell-penetrating peptide, induced more robust exon skipping in vitro and in vivo in 4^CV cells than the equivalent 2OMeAOs. As we had previously shown that a hierarchy of exon skipping efficiency induced by 2OMeAOs was also seen when the same sequences were evaluated as PMOs 46, the most effective mixtures of 2OMe and PMO AO appeared comparable in the 4^CV model, although there was a minor shift in the relative proportions of exon excised transcripts. The optimal 2OMeAO cocktails induced removal of approximately equivalent amounts of exons 52 and 52 and 53, whereas the equivalent PMO combinations generated transcripts missing exon 53 as the predominant transcript and exons 52 and 53. Approximately 10% of restored dystrophin protein was induced according to calculation of band densitrometry of western blot analysis.

PMO cocktails, consisting of a total of 40 µg of the oligomers directed to exons 52 and 53, were administered into the TAs of 4^CV mice by a single intramuscular injection. Two weeks later, dystrophin was observed by dystrophin immunofluorescence after C1, C2 or C3 treatment. Calculation of band densitometry from western blot analysis demonstrated approximately 5–10% of restored dystrophin protein after C1, C2 or C3 treatment. Further studies in this area may provide additional information to understand and to refine more efficient combinations for AO-induced dual exon skipping.

In summary, there still appears to be no consistent pattern in AO design for targeted exon skipping. Masking an obvious splice motif, such as the donor splice site that proved so effective in the mdx mouse, was ineffective in excising different exons in another mouse model of muscular dystrophy. AO cocktails were essential for by-passing the mutation in the 4^CV mouse in terms of both specificity and efficient exon removal. Consistent with other studies, the PMOs appeared to be superior to 2OMeAOs, especially in vivo. Long-term systemic treatment of the 4^CV mouse is now underway to establish an appropriate delivery regimen that would be best suited for dual exon skipping in a region of the dystrophin gene located within the human deletion hotspot.

Acknowledgements

The authors received funding from the National Institutes of Health (RO1 NS044146-02), the Muscular Dystrophy Association USA (MDA3718), the National Health and Medical Research Council of Australia (303216), and the Medical and Health Research Infrastructure Fund of Western Australia. C.M. was supported by a scholarship from Faculty of Medicine, Siriraj Hospital, Mahidol University. P.L.I. and H.M.M., who supplied the PMOs, disclose that they hold stock in a company that has an interest in developing therapeutic anti-sense oligomers. This may be perceived as a conflict of interest. We thank Russell Johnsen for technical advice and assistance with aspects of the dystrophin immunofluorescence and western blotting.