RESEARCH ARTICLE Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells A Aartsma-Rus, WE Kaman, M Bremmer-Bout, AAM Janson, JT den Dunnen, G-JB van Ommen and JCT van Deutekom Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg, AL Leiden, The Netherlands As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 2 0 -O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholino- induced skipping levels were only 5–6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficien- cies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping. Gene Therapy (2004) 11, 1391–1398. doi:10.1038/ sj.gt.3302313; Published online 1 July 2004 Keywords: antisense; exon skipping; 2 0 -O-methyl phosphorothioate; LNA; morpholino; dystrophin Introduction Antisense oligonucleotides (AONs) have been reported to modulate pre-mRNA splicing in several studies. 1 For instance, AONs have restored normal splicing by blocking cryptic splice sites, 2,3 altered the ratio of alternative splicing from malignant to nonmalignant isoforms, 4 and induced exon inclusion for mutated exons that were otherwise skipped. 5 In these studies, the AON treatments aimed at the re-establishment of wild-type mRNA. Recently, AONs have alternatively been used to restore the disrupted reading frame of dystrophin mRNAs in Duchenne muscular dystrophy (DMD) gene therapy studies. DMD patients suffer from severe muscle degeneration due to frame-disrupting mutations in the DMD gene that prema- turely abort the synthesis of the dystrophin protein. 6–9 In contrast, mutations in the DMD gene that do not affect the reading frame generate internally deleted but partly functional dystrophins and result in less severe Becker muscular dystrophy (BMD). 10,11 AON-induced restoration of the DMD reading frame is based on inducing the skipping of specific exons. This was successfully applied in cultured muscle cells from DMD patients and in the mdx mouse model. 12–19 High exon skipping levels of up to B90% were achieved, allowing the synthesis of significant levels of BMD-like dystrophins in over 75% of treated cells. 17 These dystrophins located appropriately to the sarcolemma and restored the dystrophin–glycoprotein complex, a strong indication of functional restoration. The AONs used in these studies contained 2 0 -O- methyl modified ribose molecules to render them RNase- H independent, and a full-length 2 0 -O-methyl phosphor- othioate backbone (2OMePS AONs) (Table 1). Although 2OMePS AONs have advantages such as increased resistance to nuclease degradation and increased uptake when compared to phosphodiester AONs, disadvan- tages are that a phosphorothioate backbone is to some extent cytotoxic, and may elicit an immunogenic re- sponse. 20,21 Recent developments in oligochemistry have provided AONs with different biophysical, biochemical and biological properties based on various modifications to the sugar or the backbone of the nucleotides. Modified AON analogs include morpholino-phosphorodiamidates (morpholinos), locked nucleic acids (LNAs) and peptide nucleic acids (PNAs) (reviewed by Manoharan 21 ). In morpholinos, the sugar phosphate backbone of DNA is replaced by morpholino-phosphorodiamidate oligonucleotides (Table 1). 22,23 Morpholinos are nontoxic, nuclease resistant, 23 have an increased affinity for RNA and are suggested to disrupt the secondary structure of Received 29 January 2004; accepted 25 April 2004; published online 1 July 2004 Correspondence: Dr JCT van Deutekom, Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Gene Therapy (2004) 11, 1391–1398 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00 www.nature.com/gt