Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides
Introduction
Mutations in the human dystrophin gene are responsible for Duchenne muscular dystrophy (DMD) and the less severe, allelic form of Becker muscular dystrophy (BMD) [1]. The reading-frame hypothesis (as shown below) has supplied a molecular distinction between DMD and BMD phenotypes for the majority of cases [2]. Nonsense mutations, or genomic deletions/duplications which disrupt the reading frame of the dystrophin mRNA, lead to premature termination of dystrophin protein synthesis and the DMD pathology. In contrast, BMD mutations are generally found to be in-frame deletions where the reading frame is maintained, resulting in the synthesis of shorter but semi-functional dystrophin proteins.
The mdx mouse is an animal model of Duchenne muscular dystrophy [3]. It has been used to test therapies such as myoblast transfer and the introduction of a functional dystrophin gene, either directly in a plasmid or through a viral vector 4, 5. A nonsense mutation at base 3185 of the dystrophin gene transcript inactivates this gene in the mdx mouse, resulting in termination of translation within exon 23 [6]. No functional dystrophin should be expressed in mdx muscle but immunohistochemical staining has been used to detect rare, dystrophin positive-fibres (typically less than 1%) in mdx muscle tissue [7]. These fibres (called 'revertants' as they have reverted back to the normal dystrophin-positive state) have also been detected in many DMD patients 8, 9. It has been proposed that the dystrophin in these revertant fibres arises from an exon-skipping mechanism 10, 11. In the mdx example, alternatively-processed in-frame dystrophin gene transcripts skipping the exon carrying the primary nonsense mutation have been identified [12].
Antisense oligonucleotides (AOs) have been applied to down-regulate gene expression by blocking translation [13]or by targeting an RNA for RNaseH degradation [14]. AOs have also been used to target specific regions of DNA to inhibit transcription by RNA polymerase II 15, 16. In yet another approach, phosphorothioate 2′-O-methyl oligoribonucleotides, which do not induce RNaseH activity when bound to the RNA target, have been used to restore correct splicing in a thalassemic pre-mRNA by blocking aberrant splice sites 17, 18.
The huge dystrophin gene spans 2.4 Mb and consists of 79 exons which must undergo extensive pre-mRNA processing to produce the mature mRNA of 14 000 bases [19]. Although induction of specific genomic deletions does not seem currently practicable, the potential exists for intervention during the processing of the dystrophin pre-mRNA. Two other groups have already demonstrated the very different applications of AOs to re-direct dystrophin gene transcript processing. Pramono et al. induced efficient skipping of dystrophin exon 19 in normal human lymphoblastoid cells by directing AOs to the exon recognition site of exon 19 [20]. Although expression was at very low levels in these cells, the dystrophin Kobe mRNA (skipping exon 19) was produced from a normal intact dystrophin gene. Rather than inducing a disease-associated dystrophin mRNA, Dunckley et al. used AOs directed at the splice sites flanking exon 23 of the mdx dystrophin gene to remove this exon (and the mdx nonsense mutation) from the mature mRNA [21]. The AOs used by this group did not appear to be efficient or specific as only very low levels of one alternatively-processed dystrophin transcript (with skipping of exons 22–29) were detected.
We describe an independent study of the application of AOs to induce efficient skipping of exon 23 during the processing of the mdx pre-mRNA in cultured myotubes. An mdx dystrophin gene transcript skipping exon 23 would be in-frame and could thus be translated into a slightly shorter dystrophin. As outlined below, an AO-based approach could also be used to induce skipping of one or more exons that flank genomic deletions in order to restore the reading frame of a DMD mRNA so that the phenotypic consequences of serious mutations in the dystrophin gene could be minimized. If such a therapeutic approach could be efficiently applied, a boy with a DMD genotype (e.g. 'DYS' nonsense or 'DYSxyz' frameshift mutations) may only develop a milder 'DYSROPHIN' Becker phenotype.
Section snippets
Splice site characterization
DNA was prepared from normal and mdx mouse liver samples using the DNA Direct kit (Dynal). All oligonucleotides used in this study, either for PCR, sequencing or antisense studies, are shown in Table 1.
Long range PCR (Perkin Elmer rTth XL) was carried out across dystrophin introns 22 and 23 using primers based on the dystrophin cDNA sequence (primers Ex22F and 24R). PCR products were purified from unreacted primers and nucleotides using QIAquick PCR spin columns (Qiagen) according to the
Characterization of dystrophin splice sites and design of AOs
Amplification of genomic DNA using primers Ex22F and 24R generated a single product of approximately 4.0 kb. Introns 22 and 23 were found to be 900 and 2850 bp long, respectively. Limited DNA sequencing of this material identified the splice sites flanking introns 22 and 23 (Fig. 1). GenBank accession numbers are AFO62828, AFO62829, AFO62830 and AFO62831. Rodent splice site efficiencies were calculated [22]. Characterization of these splice sites allowed the design of 2′-O-methyl dys 3′ and dys
Discussion
DMD and BMD are allelic X-linked diseases which affect about 1 in 3500 live male births where the severe, early-onset DMD cases are more frequent than the milder BMD patients. DMD is typically associated with less than 3% of normal dystrophin expression levels while BMD is seen with dystrophin levels of 10% or more [24]. The presence of any dystrophin, albeit of reduced quality and/or quantity in BMD patients, highlights the significance and ability of a shortened but semi-functional dystrophin
Acknowledgements
This work was supported by the Muscular Dystrophy Association of Western Australia, the Neuromuscular Foundation of Western Australia Inc. and grants from the Muscular Dystrophy Association of USA (to S.D.W.). R.K. was supported by grants from the National Institutes of Health, USA. We thank Dr. Nigel Laing and Professor Terry Partridge for helpful discussions and suggestions during this work.
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