Acta Myologica • 2007; XXVI; p. 179-184
Optimizing exon skipping therapies for DMD T. Yokota, W. Duddy, T. Partridge Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, USA Exon skipping is one of the more promising therapeutic options for Duchenne Muscular Dystrophy (DMD). The idea is to use antisense oligonucleotides to splice out selected exons from the pre-mRNA, at or next to the mutation site, so as to generate a translatable transcript from the mutant dystrophin gene. In principle, the majority of DMD mutations can be rescued by targeting selected exons. Recent developments of antisense oligonucleotides (AOs) such as 2`O-methylated antisense oligonucleotides (2OMeAOs) or phosphorodiamidate morpholino oligomers (morpholinos, PMOs) have made it possible to restore dystrophin expression body-wide in dystrophic mice and dystrophic dogs by single or multi-exon skipping with no obvious side-effect. Since such treatment would, in many cases, require bespoke design of AOs, it is important to demonstrate treatment of a variety of mutations in dystrophic animals. In-frame deletion patterns usually result in a mix of Duchenne and milder Becker Muscular Dystrophy (BMD), but the ratio of Duchenne to Becker varies between patterns, and this provides useful information for selection of the exons that might most profitably be targeted. This review summarizes recent progress in exon skipping therapy and discusses future strategies. Key words: Duchenne/Becker muscular dystrophy, dystrophin, revertant fibers, morpholinos
Duchenne muscular dystrophy and Becker muscular dystrophy Duchenne muscular dystrophy (DMD) is one of the more common lethal inherited diseases with an incidence of 1 in 3,500 live male births worldwide. The gene mutated in DMD and milder Becker muscular dystrophy (BMD), encoding the dystrophin protein (1), is the largest known gene, with 79 exons spanning 2.4 Mb of DNA. Disease phenotype varies considerably. In-frame mutations are commonly associated with BMD phenotypes ranging from almost asymptomatic to severe DMD-like forms. In contrast, out-of-frame mutations show a strong association with severe DMD phenotypes. This “reading frame rule” holds true for 92% of DMD/BMD cases. For instance, in-frame deletion of exon 16 is not associated with disease (2). Interestingly, the length of deleted area in the gene is not the most important factor in determining clinical phenotype. For example, deletion of exons 45-55, spanning 11 exons, is associated with disease severities ranging from mild to almost asymptomatic dystrophy
(3). The middle portion (rod domain) of dystrophin (exons 10-60) has been noted to accommodate large deletions without serious phenotypic consequence. The most dramatic example is an in-frame deletion of exons 17-48 (46% of the gene), which was discovered in a very mild case of BMD (4).
Dystrophin revertant fibers and exon skipping mechanisms Spontaneous restoration of reading-frame of dystrophin mRNA by exon-skipping actually occurs in many DMD patients and animal models. Scattered through dystrophic muscle are occasional foci of “revertant” fibers strongly positive for dystrophin which increase in number during cycles of degeneration/regeneration (5, 6). Expression of dystrophin in these “revertant” fibers results from spontaneous skipping of exons around the primary mutation (7). Such spontaneous exon skipping of diseasecausing nonsense mutations occurs in several genes such as the Factor VIII gene in hemophilia, the fibrillin gene in Marfan syndrome and the ornithine d-aminotransferase gene in gyrate atrophy (8, 9). However, in these cases, it has not been established whether the facility to skip exons is focal and clonal as it is in dystrophic muscle. Dystrophin-positive fibers generated by AOs gradually disappear within a month or so after AO injection, but spontaneous “revertant” fibers persist for some years and increase with age (6). This expansion of “revertant” fibers appears to reflect the protective effect of the dystrophin they contain, but the mechanism whereby each clone consistently and efficiently skips specific exons is not currently understood. Such an understanding might aid the development of long-term AO-induced exon skipping therapy.
Development of a new generation of antisense oligos Various backbone chemistries of antisense oligonucleotide have been tested to overcome the problems of in vivo breakdown of DNA or RNA. Recent explorations of drug-like characteristics of AOs have lead to the development of oligonucleotides that contain phosphorothioate linkages throughout their length and 2’O-modifications of
Address for correspondence: Toshifumi Yokota, Center for Genetic Medicine Research, Children’s National Medical Center, 111 Michigan Ave, NW, Washington, DC, 20010, USA. Fax: +1 202 4766014. E-mail:
[email protected]
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the ribose moiety (e.g. 2’-O-methyl, 2’-O-methoxyethyl). Previously we have shown that intramuscular injection of 2’-O-methyl antisense oligonucleotides (2oMeAO) can restore dystrophin expression (10). When systemically delivered, it did generate dystrophin in body-wide skeletal muscles of mdx mice, but only marginal amounts (11). A clinical trial in The Netherlands involves intramuscular injection of 2OMeAOs (P-S) into the TA muscle of patients with mutations correctable by exon 51 skipping. Phosphorodiamidate Morpholino Oligomers (Morpholinos, PMOs) have a number of additional advantages over other chemistries, such as high water solubility. Furthermore, morpholinos are not subject to metabolic degradation, do not activate toll-like receptors and do not activate the interferon system or the NF-(kappa)B mediated inflammation response (12). Recently, we have shown that systemic injections of PMOs can restore dystrophin production to functional levels in many muscles of the mdx mouse and ameliorate dystrophic pathology without any trace of toxicity (13). This approach is currently being tested in DMD dogs with similarly encouraging results (Yokota et al., unpublished observations). A clinical trial, planned in the UK, proposes to locally inject a 30 mer of single morpholino, targeting the Exonic splicing enhancer (ESE) sequence of exon 51. They will inject three different concentrations (low, intermediate and high – 2 boys per concentration), into extensor digitorum brevis and analyze the biopsy one month after injection (14). Development of a new AO drug is also underway. Recently, Wilton et al. reported that peptide tagged morpholinos show much greater efficiency than untagged bare morpholinos (15). However, they also showed elevated blood urea nitrogen (BUN) after injection into mice, indicative of toxicity. Whether or not tagged PMOs are better than non-tagged AO drugs will depend on the balance between increased efficacy and increased toxicity. Attention must also be paid to the question of whether there is any immune response in the long term to the peptide tag.
Animal models to test exon skipping Conventionally, the mdx mouse model has been much used for animal research on DMD. The dystrophin defect arises from a nonsense mutation in exon 23. Both 2OMeAO and morpholinos (11, 13) against exon 23 have been shown to efficiently skip the exon and restore dystrophin expression in mdx mice. However, the same mutation is very rare in humans, there being no reports of it in the Leiden Muscular Dystrophy database (http://www. dmd.nl) (16), so exon 23 will not be a target in any early human trials. In man, most DMD mutations are deletions, with a lesser number of duplications, that compromise the open reading frame. Of deletions, 80% begin and end within the rod domain of the dystrophin gene and 90% of these occur within a “hotspot” region, from exons 42
to 57. At least two mutant mice harbor mutations in this region, mdx52, where exon 52 is lacking, and mdx-4cv with a nonsense mutation in exon 53. Both will be useful for testing the feasibility of AOs (17, 18) targeted at regions of interest for therapy in man. AOs targeting exon 51 or exon 53 can restore the mdx52 mutation, and dual targeting of exon 52 and exon 53 can restore the mdx4cv mutation. More interestingly, both mice can also be used as test-beds for multi-exon skipping, targeting for example, exon 45-55 (discussed in following chapter). A further aspect of interest in mutations in this area of the dystrophin gene is the fact that they disrupt the production of the dp260 isoform of dystrophin that is expressed in the retina (19), and it is possible to determine whether AOs that restore reading frame in these mice are effective in the appropriate retinal layer. Mdx-3cv is also an interesting animal model to test exon skipping because it is the only mouse model with a mutation in the cysteine rich domain (20) due to a deletion within intron 65. This alters mRNA processing such that several transcripts are produced, predominantly one lacking the whole exon of exon 66. AOs targeting exon 65 (and 66) could result in exon skipped products with restored reading frame. Importantly, the cysteine rich domain is responsible for dystroglycan binding, so it would be interesting to test whether dystrophin lacking this region can ameliorate the Mdx-3cv phenotypes. Additionally, the dystrophin isoform Dp71, which among known dystrophic animals is lacking only in mdx-3cv, is thought to play an important role in brain. Haenggy et al. (21) previously investigated mice lacking either utrophin (utrophin0/0) or dystrophin isoforms (including Dp71) (i.e. mdx3Cv), and found three distinct complexes: (i) DAPs associated with utrophin in the basolateral membrane of the choroid plexus epithelium; (ii) DAPs associated with utrophin in vascular endothelial cells; and (iii) DAPs associated with Dp71 in the glial end-feet. The composition and localization of the Dystrophin associated proteins (DAPs) are dependent upon the anchoring proteins. Upon ablation of utrophin or Dp71, the corresponding DAPs were disrupted and no compensation of the missing protein by its homologue was observed. Association of the water channel aquaporin-4 with the glial DAPs likewise was also disrupted in mdx3Cv mice (21). Aquaporin-4 is known to be localized by alpha1-syntrophin, a dystrophin associated protein, at glial astrocyte endfeet, and involved in generation of cerebrospinal fluid (CSF) and brain edema (22). Thus, restoration of Dp71 by AOs could localize DAPs, including alpha1-syntrophin which is associated with the C-terminal domain of Dp71, then, in turn, restore the localization of aquaporin-4 at the blood-brain barrier (BBB) (23). Importantly, AO sequences against the equivalent dystrophin region in human and animal models (mouse or dog) show few differences across species. Development of gene-modified mice, possessing the human instead of mouse dystrophin gene has proved a
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useful tool to test the efficacy of AO sequences in vivo as demonstrated by Arechavala-Gomeza et al. for comparative analysis targeting exon 51 (24).
ping can be achieved (Table 1). Multi-exon skipping has been demonstrated in vivo in mdx mice (25) and dystrophic dogs (Yokota et al., unpublished observation). Interestingly, deletion of exons 45-55 is associated with a milder phenotype than other smaller in-frame deletions within the exon 45-55 range (Table 2) (26). Therefore, multiexon skipping targeting exon 45-55 may well ameliorate the clinical phenotype of patients with in-frame deletions within this region, whether DMD or BMD. In this context, the population of patients for whom exon skipping therapy is appropriate is probably larger than formally estimated. Even when patients would be theoretically treatable by single exon skipping, multi-exon skipping may well be a better option if the resulting truncated protein is
DMD patient population applicable for exon skipping therapy The goal of exon skipping therapy is generally to change the mutations of DMD patients from out-of-frame to in-frame. In this context, we can estimate the number of patients applicable for exon skipping therapy from the Leiden database (http://www.dmd.nl) (16). It is estimated that around 70% of patients with deletions can be treated by single exon skipping, rising to 90% if multi-exon skip-
Table 1. Single exon skipping vs. multi-exon skipping for DMD patients with dystrophin deletions. No. patients
Single exon skipping
Multi-exon skipping
208
275
Exon skipping potentially applicable Not applicable
#
Applicable patients
95
28
69%
90%
Only patients reported from the United States (Columbus) are included (303 in total). Numbers for each deletion pattern and the number of applicable patients are taken from the Leiden Muscular Dystrophy database (http://www.dmd.nl) and our previous report (26). Criteria for the capability of exon skipping is based on the previous paper (26). # In some patients, deletions are in-frame so exon skipping is not applicable in a frame-correction capacity, however, exon skipping may still be applicable for modification of defective protein.
Table 2. In-frame deletions within hotspot region of rod domain grouped according to common attributes. Description of set of in-frame deletion patterns
No. DMD
No. BMD
DMD %
Start from exon 45
53
235
18
Start from exon 47
13
10
57
Start from exon 48
24
25
49
Start from exon 49
11
1
92
Start from exon 50
9
1
90
End at exon 46
2
9
18
End at exon 47
20
95
17
End at exon 48
17
78
18
End at exon 49
11
40
22
End at exon 51
31
22
58
End at exon 53
25
12
68
End at exon 55
5
16
24
Include all or part of hinge 3#
68
50
58
Do not include hinge 3
55
222
20
Create a possible hybrid STR
58
121
32
Join STR ends
10
10
50
Leave fractional STRs
55
141
28
#
Numbers of in-frame mutation patterns within ex42-57 were calculated by combining data from selected countries in the Leiden Muscular Dystrophy database, as described in main text. A deletion was considered to include all or part of hinge 3 if it included exons 50 and/or 51. Possible hybrid Spectrin-Type Repeats (STRs) are described in the main text and diagrammatically in Figure 1. Deletions starting from exons 42-44, 46, 51, 52-56, or ending at exons 42-45, 52, 54, 56-57, are not presented separately as they number five or less, but are still included among the totals relating to hinge 3 and STRs. # In-frame deletions including all or part of hinge 3 (Ex50/51) lead to significantly more DMD than in-frame deletions excluding hinge 3 (by chi square test, p < 0.005).
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more functional. This “multi-exon skipping” strategy is likely to be attractive to pharmaceutical companies since the oligo cocktail can be regarded as “a single drug”, requiring only a single toxicology study. And, as suggested recently by Beroud et al., multi-exon skipping of exon 45-55 could rescue up to 63% of DMD patients (3). In addition, exon skipping for duplication was recently demonstrated in human cells (27), and around 80% of DMD cases with duplication mutations are also potentially treatable (25). A recent report by Kesari et al. using MLPA analysis, indicates that the population of BMD with duplication mutations is higher than previously expected, suggesting that in-frame duplications often yield partially functional dystrophin protein, and, therefore, that many out-of-frame duplications may be amenable to the exon skipping approach by targeting only a part of the duplicated region (Kesari et al., unpublished observations). Similarly, a considerable number of patients with splice site mutations could also be treated with AOs. For example, a dog model of DMD, Golden Retriever Muscular Dystrophy (GRMD) or Canine X-linked Muscular Dystrophy (CXMD) harbors a mutation in intron 6, which leads to the loss of exon 7 from mRNA. We have recently shown that a cocktail of morpholinos targeting exon 6 and exon 8 can restore reading frame and dystrophin expression body-wide after systemic injections (Yokota et al., unpublished observations). Exon skipping therapy could also be applicable for many other types of mutation such as small deletions/insertions, missense mutations, and more complicated rearrangements, although extent of functional recovery after exon skipping might vary among targeted exons since some in-frame mutations lead to DMD rather than BMD, in contravention to the reading frame rule (discussed below).
Is reading frame rule everything? Most deletions (80%) begin and end within the rod domain of the dystrophin gene, of these 90% occur within the “hotspot” region, from exons 42 to 57. Structurally, this region encodes seven spectrin-type repeats (STRs; numbered 16 to 22) and the “hinge 3” region between STR 19 and STR 20 (Fig. 1). Cases reported in the Leiden Muscular Dystrophy database (http://www.dmd.nl) for deletions within the hotspot region are now sufficiently numerous that differences in the Duchenne/Becker ratio are often statistically significant between in-frame deletion patterns, especially if one permits the grouping together of deletion patterns with common attributes such as the exon they start or end at. We combined data from reports (from Argentina, Belgium, Brazil, Bulgaria, Canada, China, Denmark, France, India, Italy, Japan, The Netherlands, UK, and USA) as of February, 2007, where diagnoses were performed using MLPA/MAPH, southern blotting, or PCR primer sets that allow deletion boundaries to be assigned accurately to a specific exon (Fig. 1, Table
2). It is true that some mutations may have been mapped incorrectly, and that differences in diagnostic criteria of DMD/BMD between sites/countries may have introduced some inconsistencies into the database that will appear as “noise”. But there is no reason to suspect any systematic bias and general tendencies will remain detectable with large data sets. In-frame deletion patterns, even within the rod domain, usually result in a mix of Duchenne and Becker (Table 2), and interestingly, the ratio of Duchenne to Becker remarkably varies between patterns. For example, in-frame deletions of ex47-51, ex48-51, and ex49-53 are reported to be associated with DMD (28, 29) (Fig. 1). Likely contributor factors to these differences include stability or function of truncated protein structure, the effect of the deletion on alternative splicing, and translation/transcription efficiency after genome rearrangement. Spectrin-type repeats (STRs) in the rod domain are bundles of three alpha-helices that are unlikely to form stable structure unless complete. However, Sadaart et al. (30) and Menhart (31) have shown that the hybrid STR, resulting from deletion of exons 41 and 42, is stable in vitro, and have proposed general principles, illustrated in Figure 1 to identify: i) hybrid STRs, ii) deletions that join STR ends together, and iii) deletions resulting in fractional, therefore misfolded, STRs (30, 31). Deletions that join STR ends are not common but the few that do occur are more commonly associated with DMD than possible “hybrid STRs” (Table 2), supporting Menhart’s suggestion that they are less functional than hybrid STRs. On the other hand, the database survey reveals that deletions leaving “fractional STRs” are more commonly associated with BMD than those yielding predicted possible “hybrid STRs”, contrary to our expectations (Table 2). Two deletion patterns strongly associated with mild clinical phenotype (ex45-47 and ex45-49) make up more than half the cases in which fractional STRs are predicted. It would seem, therefore, that we must look for other features of these deletions to account for the severity of disease with which they are associated. Besides the primary binding site of F-actin in the N-terminus of Dystrophin, actin also binds the rod domain between STRs 11 to 17 (encoded by ex31-45) (32, 33). It is not known whether different deletion patterns differentially affect actin binding, however, it seems that the presence/absence of the actin-binding site does not correlate simply with the different phenotypes in each pattern in the database, since deletions starting from exon 45 result in milder phenotypes than those starting from later exons (Table 2). A more general rule is that deletions starting from exon 45 and ending before hinge 3 tend to be associated with mild phenotypes irrespective of the predicted STR structure (Table 2). It is also noteworthy that, exon 4555 deletions, which remove hinge 3 are strongly associated with mild clinical phenotype (3) but deletions whose breakpoints fall adjacent to hinge 3, such as D50/51, or D49-53 usually lead to DMD rather than BMD. This co-
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Figure 1. Diagrammatic representations of dystrophin structure showing spectrin-type repeats (STRs) for in-frame rod domain deletions within hotspot. Exons 42 to 57 of the dystrophin gene, encoding STRs 16 to 22, are represented at top, with the hinge 3 region labeled. Exon boundaries are represented by vertical lines, left-facing arrows, and right-facing arrows, indicating that the next exon begins at, respectively, the first, second, or third, nucleotide of a codon. STRs are shaded differently and STR labels are in black or white for easier reading. Truncated structures for each deletion pattern are represented, together with a description of the new structure resulting at each join, and the respective numbers of Duchenne and Becker cases. Some truncated structures may contain correctly folded ‘hybrid’ STRs. Deletions having less than ten cases are listed last, without associated diagrams. Numbers were calculated by combining data from selected countries in the Leiden Muscular Dystrophy database, as described in text.
nundrum is not readily explicable, but it might suggest that there are limits on the size of the rod domain if the hinge domain is lacking, perhaps because hinge domains are needed to give flexibility to rod structures beyond a certain length (34). Clearly, we need a better understanding of these differences if we are to make rational choices of exon-skipping targets that will generate the most functionally effective “quasi-dystrophins”. In this quest for a better understanding of the relevant factors, perhaps the most relevant evidence is to be found in a detailed study of the genotype/ phenotype correlations in the human DMD and BMD populations. This requires the assembly of a reliable database founded on the unison of strictly defined clinical criteria with comprehensive and detailed information on the nature of the mutation ideally including the genomic, and the transcript data, and, where applicable, the exons represented in any partial dystrophin protein produced. It is encouraging to note that a number of databases approaching this level of detail are currently being organized.
Acknowledgements Authors thank Drs Akinori Nakamura, Shin-ichi Takeda, and Eric Hoffman for useful discussions.
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