Site-directed gene repair of the dystrophin gene ... - Semantic Scholar

Human Molecular Genetics, 2010, Vol. 19, No. 16 doi:10.1093/hmg/ddq235 Advance Access published on June 11, 2010

3266–3281

Site-directed gene repair of the dystrophin gene mediated by PNA– ssODNs Refik Kayali, Frederic Bury, McIver Ballard and Carmen Bertoni ∗ Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA

Permanent correction of gene defects is an appealing approach to the treatment of genetic disorders. The use of single-stranded oligodeoxynucleotides (ssODNs) has been demonstrated to induce single-point mutations in the dystrophin gene and to restore dystrophin expression in the skeletal muscle of models of Duchenne muscular dystrophy (DMD). Here we show that ssODNs made of peptide nucleic acids (PNA –ssODNs) can achieve gene repair frequencies more than 10-fold higher than those obtained using an older generation of targeting oligonucleotides. Correction was demonstrated in muscles cells isolated from mdx 5cv mice and was stably inherited over time. Direct intramuscular injection of PNA – ssODNs targeting the mdx 5cv mutation resulted in a significant increase in dystrophin-positive fibers when compared with muscles that received the ssODNs designed to correct the dystrophin gene but made of unmodified bases. Correction was demonstrated at both the mRNA and the DNA levels using quantitative PCR and was confirmed by direct sequencing of amplification products. Analysis at the protein level demonstrated expression of fulllength dystrophin in vitro as well as in vivo. These results demonstrate that oligonucleotides promoting strand invasion in the DNA double helix can significantly enhance gene repair frequencies of the dystrophin gene. The use of PNA – ssODNs has important implications in terms of both efficacy and duration of the repair process in muscles and may have a role in advancing the treatment of DMD.

INTRODUCTION Defects in the gene that encodes for dystrophin are responsible for Duchenne muscular dystrophy (DMD), a severe, X-linked recessive disorder affecting 1 in every 3500 newborn males. To date, corticosteroids are the only available treatment option to delay the progression of the disease, but this treatment does not arrest the degenerative process. Gene therapy continues to provide the best option for a cure for DMD. Several approaches have been investigated in the attempt to restore dystrophin expression in skeletal muscles. Among the gene therapy applications currently under investigation for DMD, viral vectors have been shown to be the most effective (1,2). The use of viral-mediated technologies, however, is limited by several hurdles, most significantly the size limitation of the gene that viral capsid can host and the immunological responses associated with the use of some of those viral vectors. An alternative approach to gene augmentation strategies is the use of technologies aimed at restoring

dystrophin expression by correcting the genetic defect (3). The use of antisense oligonucleotides to redirect mRNA splicing of dystrophin transcripts and to restore its expression in the skeletal muscles has gained attention in recent years, thanks to the promising results obtained by several laboratories around the world (4 – 9). However, the amount of dystrophin protein being restored is limited and the effects achieved only temporary. In addition to antisense-mediated therapies, oligonucleotides can be used to induce single base pair alterations at the genomic level. This technology takes advantage of innate cellular repair mechanisms that maintain chromosome stability. Introduction of oligonucleotides containing one or more mismatches in the cell nuclei is known to activate these repair mechanisms and induce the desired correction (10). The initial studies of gene repair for DMD were conducted using a chimeric oligonucleotide (chODN) designed to target the single-point mutation in the mdx mouse (11,12). Additional findings from an independent group demonstrated the feasibility

∗

To whom correspondence should be addressed at: Department of Neurology, University of California at Los Angeles, 710 Westwood Plaza, Los Angeles, CA 90095, USA. Tel/Fax: +1 3108256387; Email: [email protected]

# The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Downloaded from http://hmg.oxfordjournals.org at University of Texas at Dallas on July 22, 2010

Received December 4, 2009; Revised May 4, 2010; Accepted June 7, 2010

Human Molecular Genetics, 2010, Vol. 19, No. 16

Several studies have also demonstrated that gene repair can be enhanced by synchronizing the cells in the S phase of the cell cycle or by reducing the rate of replication fork progression (20,21,26,27). These observations have prompted us to investigate whether ssODNs made of peptide nucleic acids (PNAs) bases can promote the conversion of a single base at the genomic level and restore dystrophin expression in skeletal muscles of mdx 5cv mice. PNAs are DNA mimics with a pseudopeptide backbone able to form stable duplex structures with Watson –Crick complementary DNA, RNA (or PNA) oligomers to promote helix invasion in duplex DNA. Furthermore, the binding affinity of PNAs to DNA targets is stronger than that of DNA/DNA interaction. This enhanced binding affinity is partially due to the uncharged property of the PNAs that diminishes the electrostatic repulsion formed from DNA/ DNA duplexes. As a result, the length of oligonucleotides necessary to achieve maximal effects is also significantly reduced. In most of the hybridization studies, PNAs with lengths of 12– 18 nucleotides are sufficient to form strong duplex formation and to distinguish single base mutations. In this report, we show that PNA – ssODNs can induce stable single base pair alterations in the dystrophin gene and result in a significant increase in the level of dystrophin being expressed when compared with oligonucleotides made of unmodified bases. The use of PNA oligonucleotides in vivo corrected the dystrophin gene defect in post-mitotic nuclei leading to sustained expression of dystrophin for up to 4 months after injection. Furthermore, PNA – ssODNs restored expression of dystrophin throughout the entire length of the fiber in a subset of myofibers. These data demonstrate that gene correction in skeletal muscles can be increased by facilitating the interaction of the ssODNs with their target and by promoting DNA strand invasion of the targeted genomic sequence. The use of PNA – ssODNs will have important clinical implications in the development of an effective treatment to muscle diseases and in particular DMD.

RESULTS Design and pairing targeting the mdx 5cv mutation The mdx 5cv model is characterized by a single base pair mutation (an A-to-T transversion) in exon 10 of the dystrophin gene that generates a cryptic splice at this position (Fig. 1A). As a result, the dystrophin mRNA is aberrantly spliced 53 bp upstream the normal dystrophin exon 10/intron 10 splice junction causing a shift in the reading frame with a premature stop codon, a few amino acids downstream the mdx 5cv mutation and complete absence of dystrophin protein. Each targeting oligonucleotide was designed to induce a T-to-A transversion to correct the mutation of the dystrophin gene in the mdx 5cv strain (Fig. 1B). The targeting ssODNs were designed to be homologous to the transcribed or coding strand of the gene (CORC and PNA – CORC) or complementary to it (CORNC and PNA – CORNC). The ssODNs made of regular bases have been described previously (14). They shared a 45 bp identity to the mdx 5cv gene and contained a fluorescent label modification (CY3) at their 5′ ends and four unmatched phosphorothioate DNA bases at their 3′ ends


of oligonucleotides to correct the mutation in the GRMD (13), a dog model for DMD and expanded the applicability of oligonucleotide-mediated gene editing to larger models for the disease. During the past years, considerable progress in the field of gene correction for muscular dystrophy has been made (14). We have expanded the therapeutic potentials of chODN by targeting intron/exon boundaries of the dystrophin gene to induce skipping of the exon causing the mutation, an approach that can treat the majority of deletions and frameshift mutations causing DMD (15). We have also demonstrated that singlestranded oligodeoxynucleotides (ssODNs) are as efficient as chODN in inducing gene correction (14). The lower cost of production and the possibility to scale up the synthesis of oligonucleotide have made this technology a realistic possibility for the treatment of the disease. In myoblasts, frequencies of gene repair differ largely, depending on the type of mismatch introduced on the oligonucleotide and the strand of the genomic DNA being targeted. For instance, ssODNs designed to alter a single base pair on the coding strand of the genomic DNA were shown to be more efficient than ssODNs designed to target the non-coding strand (14). Those results are consistent with those previously reported by several independent groups and appear to be common to both prokaryotic and eukaryotic systems (16– 18). Correction in myoblasts appear to be sustained over prolonged periods of time, as demonstrated by the ability of cells that have undergone repair to express dystrophin upon induction of differentiation after being maintained in culture for up to 2 weeks after ssODN transfection (14,19). The mechanisms for gene repair mediated by ssODNs are still largely unknown. To date, gene targeting has been studied in bacteria, yeast and a variety of mammalian cell lines. Those reports have implicated homologous recombination as one of the major mechanisms that influence gene repair mediated by oligonucleotides, although in certain mammalian cells, nucleotide exchange repair and double-strand break repair have also been suggested as potential mechanisms involved in single base pair exchange between the oligonucleotide and the DNA (20– 22). We have recently demonstrated that the use of ssODNs containing specific modifications capable of activating the base excision repair (BER) mechanism can profoundly influence the frequencies of gene repair mediated by oligonucleotides in myoblasts, suggesting that in muscle cells, repair mechanisms other then homologous recombination can actively participate in gene repair (19). Despite promising results for gene therapy, however, the frequencies of gene repair remain too low to be clinically applicable in Duchenne patients. The limiting step in the repair process in myoblasts does not appear to be the delivery of ssODNs as oligonucleotides are readily taken up in cells in culture (12,19). The low efficiencies are thought to be related to the inability of the oligonucleotides, once inside the cells, to reach their target genomic region. This hypothesis is supported by the observation that increasing the stability of the oligonucleotides and facilitating their interaction with the genomic DNA targeted for repair markedly enhance the frequencies of gene repair induced by ssODNs. For example, ssODNs containing 2′ -O-methyl RNA residues (16,23), phosphorothioate (PS) linkages (18,24) or locked nucleic acid (LNA) bases (25) at their extremities can increase the targeting frequencies.

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(indicated as ‘tag’ in Fig. 1B). The targeting and control PNA – ssODNs shared an 18 bp homology with the sequence targeted for repair, except for the single base mismatch in the correcting PNA. Transfection efficiencies of ssODNs Oligonucleotide uptake was assessed in myoblasts transfected with a targeting ssODN or a CY-3-labeled PNA– ssODN for up to a week after transfection. Fluorescence was barely detectable in cells when the PNA – ssODN was added directly to the media, but was increased by the use of the transfection reagent LipofectamineTM 2000 (Supplementary Material, Fig. 1). Fluorescence was detected in nearly 100% of the cells in culture treated with oligonucleotides that had been complexed with the transfection reagent. Intensity was higher in myoblasts transfected with the ssODN made of unmodified bases when compared with cells treated with the PNA – ssODNs as demonstrated by fluorescent analysis (Supplementary Material, Fig. 2A) and by flow cytometry (FACS) analysis (Supplementary Material, Fig. 2B). Fluorescence remained stable for up to 48 h after transfection and then steadily declined

(Supplementary Material, Fig. 2C). As early as 72 h after transfection, there was a smaller number of CY3-positive cells in myoblasts treated with PNA – ssODNs compared with unmodified ssODNs. By 5 days, fluorescence had almost disappeared from PNA – ssODN-transfected cells while it was still detectable in cells treated with ssODNs. Dystrophin protein expression in oligonucleotide-treated mdx 5cv muscle cells Western blot analysis of mdx 5cv cells treated with targeting oligonucleotides was performed 2 weeks after transfection and 72 h after induction of differentiation (12,14,15). As positive control, we used proteins isolated from wild-type (C57Bl6/J) myotubes and maintained in differentiation media for the same period of time, although only one-tenth of the protein was loaded in the gel (35 mg). The expression of full-length dystrophin was detected in cells treated with targeting ssODNs and targeting PNA – ssODNs, but not in cells treated with control oligonucleotides (Fig. 2A). Muscle precursor cells transfected with the ssODNs designed to anneal to the non-transcribed strand of the dystrophin gene had


Figure 1. Oligonucleotide design and targeting of the mdx 5cv mutation. (A) The A-to-T mutation in exon 10 of the dystrophin gene creates an aberrant splice site in the mdx 5cv mouse. The exclusion of the last 53 bp of exon 10 leads to a frameshift in the open-reading frame of the dystrophin gene that results in the absence of dystrophin expression in this mouse model for DMD. (B) Linear sequence of targeting ssODNs (CORC, CORNC) and targeting PNA–ssODNs (PNA–CORC and PNA– CORNC). Underlined bases correspond to the single base pair mismatch between the targeting oligonucleotide and the mdx 5cv mutation. As control we used PNA– ssODNs identical to their targeting counterparts (PNA–CTLC to PNA–CORC; PNA–CTLNC to PNA–CORNC) except for the underlined base which is complementary to, rather than mismatched with, the corresponding genomic sequence. ssODNs contain a CY3 molecule at their 5′ ends and four phosphorothioate bases at their 3′ ends (indicated as ‘tag’) to increase their stability.


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higher levels of dystrophin expression than cells treated with the ssODNs targeting the transcribed strand, confirming the strand bias previously observed in muscle cells in culture (14). Cells transfected with the targeting PNA – ssODNs consistently showed higher levels of dystrophin expression than cells treated with ssODNs designed to correct the mdx 5cv mutation and made of regular bases (Fig. 2A). Immunostaining analysis confirmed the expression of dystrophin in all cells treated with targeting ssODNs, but not in cells treated with control PNA – ssODNs (Fig. 2B) or control ssODNs made of regular bases (not shown). The pattern of dystrophin protein staining in myotube cultures was similar to what we have described previously in studies of ssODN-induced dystrophin gene repair in vitro and demonstrated the presence of myotubes composed of nuclei that had undergone gene repair and non-corrected myonuclei (12,14,15). Evidence of gene correction of the mdx 5cv mutation at the genomic DNA level To confirm that the expression of dystrophin detected by western blotting and immunostaining was due to a correction of the mdx 5cv mutation, we analyzed DNA isolated from

cells treated with targeting and control oligonucleotides. The single-point mutation in the mdx 5cv mouse generates a new restriction site for HphI (Fig. 3A) (14). Digestion of total genomic DNA with HphI allowed us to cleave all copies of the mutant mdx 5cv sequence, leaving intact for amplification only that portion of DNA that had undergone single base pair alteration (refractory to HphI restriction digestion). PCRs were carried out using a forward primer homologous to a region of exon 10 upstream the mdx 5cv mutation paired to a reverse primer in intron 10 (Fig. 3B). Real-time PCR was used to monitor amplification reactions and to estimate gene correction frequencies in cells treated with targeting ssODNs and PNA –ssODNs and maintained in growth media for 1 week after transfection. Amplification was detected only in cells treated with the targeting ssODNs and the targeting PNA – ssODNs, but not in untreated cells or cells transfected with control ssODNs. PNA – oligonucleotides targeting the non-coding region of the dystrophin gene (PNA – CORC) were 3 – 5-folds more effective in inducing dystrophin gene repair than their regular counterpart (CORC) made of unmodified bases (Fig. 3C), with an overall level of correction frequencies of 3% of the total amount of dystrophin copies detected in undigested DNA. The frequencies of gene repair


Figure 2. Dystrophin protein expression in mdx 5cv cells treated with oligonucleotides. (A) Myoblasts transfected with targeting (CORC, PNA– CORC, CORNC and PNA– CORNC) or control (CORC and CORNC) oligonucleotides were maintained in culture for 2 weeks and then induced to differentiate and analyzed for the expression of dystrophin by western blot analysis and immunohistochemistry. Full-length dystrophin was detected only in cells treated with targeting ssODNs, but not in cells transfected with the control oligonucleotides (PNA– CTLC and PNA–CTLNC). The ssODNs designed to anneal to the transcribed strand of the dystrophin gene (CORNC and PNA– CORNC) showed higher level of dystrophin expression when compared with ssODN designed to anneal to the non-coding strand (CORC and PNA– CORC). Proteins isolated from wild-type myotubes were used as positive control (one-tenth the amount of what was used to detect dystrophin expression in cells treated with ssODNs). (B) Dystrophin expression was evident in mdx 5cv cells 48 h after differentiation and 2 weeks after transfection with targeting oligonucleotides. No staining was detected in cells treated with control oligonucleotides maintained in culture under similar conditions. (Scale bar: 100 mm.)

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in cells treated with the PNA – CORNC were 8 – 12-fold higher than those achieved in cells transfected with CORNC, demonstrating that 7% of the dystrophin gene had undergone gene repair and that oligonucleotides targeting the coding strand of the dystrophin gene were more effective in directing the single base pair conversion (Fig. 3D). To confirm specificity, we performed fractionation of PCR products and direct sequencing by agarose gel electrophoresis. A band of the predicted molecular weight corresponding to the exon 10/intron 10 region of the dystrophin gene was detected only in samples isolated from cells treated with targeting oligonucleotides (Fig. 3E). Direct sequencing of the 350 bp amplicons demonstrated that the correction had occurred at the genomic level, converting the mdx 5cv mutation back to the wild-type sequence (Fig. 3F).

Restoration of full-length dystrophin transcripts after gene repair As further evidence that the correction of the mdx 5cv mutation restored full-length dystrophin expression, we analyzed dystrophin transcripts in cells transfected with targeting and control oligonucleotides maintained in culture for at least 1 week after transfection and then induced to differentiate for 72 h. Total messenger RNA was reverse transcribed and amplified using primers comprising exons 10 and 11 of dystrophin (Supplementary Material, Fig. 3). All samples amplified a product of 163 bp corresponding to the aberrantly spliced mdx 5cv transcripts. Only samples isolated from cells treated with CORC, CORNC, PNA – CORC and PNA – CORNC showed an additional band of 50 bp higher molecular


Figure 3. Evidence of gene conversion mediated by PNA–ssODN at the genomic level. (A) The A-to-T transversion that characterizes the mdx 5cv mouse created a restriction site (HphI) absent in mdx 5cv myoblasts that have undergone gene repair. (B) Genomic DNA was isolated from untransfected cells or cells transfected with oligonucleotides and was subjected to overnight digestion using the restriction enzyme HphI. Following digestion, the region of the mdx 5cv mutation was PCR-amplified using primers flanking the exon 10/intron 10 region. (C and D) Gene correction was analyzed by real-time PCR using DNA isolated 1 week after transfection with control or targeting oligonucleotides and digested with HphI. Targeting oligonucleotides made of peptide nucleic acids were shown to consistently induce 5 –12-folds higher level of gene repair than unmodified ssODNs (∗ P ≤ 0.04). (E) A specific PCR product of identical molecular weight to that from wild-type cells was obtained only in cells treated with targeting oligonucleotides, but not in cells treated with control ssODNs. (F) Direct sequencing of the amplicons confirmed that the correction had occurred at the genomic level to reverse the A-to-T mutation of the mdx 5cv strain.


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weight originating from the correction of the splicing defect (Supplementary Material, Fig. 3B). Real-time PCR was used to quantify the levels of full-length transcripts generated (Fig. 4). Amplification was detected only in samples isolated from cultures of mdx 5cv cells treated with targeting oligonucleotides, but not cells treated with nontargeting ssODNs. The levels of full-length gene products were consistently higher in cells treated with PNA–CORC and PNA–CORNC when compared with ssODN targeting the mdx 5cv mutation but made of regular bases (Fig. 4B and C).

The results were consistent across multiple experiments performed and demonstrated that PNA –CORNC was more efficient in correcting the mutation in the dystrophin gene than all other targeting oligonucleotides. Full-length dystrophin expression ranged between 7 and 8% in those cells, whereas PNA – CORC was shown to restore 2 to 3% of normal dystrophin transcripts. Direct sequencing of the PCR products obtained confirmed the ability of targeting oligonucleotides to convert the T in mdx 5cv cells back into an A (not shown).


Figure 4. Restoration of dystrophin expression after oligonucleotide transfection. (A) Expression of full-length dystrophin was assessed by real-time PCR in total messenger RNA isolated from cells transfected with targeting or control oligonucleotides 1 week after transfection and 72 h after induction of differentiation. Single-stranded cDNA obtained after reverse transcription of total RNA was amplified using a reverse primer positioned in exon 10 and complementary to the region of the dystrophin mRNA spliced out in the mature mRNA and a forward primer homologous to exon 9. (B and C) The levels of dystrophin expression achieved using PNA– CORC and PNA– CORNC were quantified by real-time PCR and compared with those achieved using CORC and CORNC, respectively. No amplification was detected in untreated cells or cells treated with control PNA– ssODNs. Absolute quantities were calculated using a standard curve that was generated by mixing different amounts of total mRNA isolated from mdx 5cv and C57 cells. All amplicons were normalized to GAPDH (∗ P ≤ 0.05). (D) Amplicons obtained from real-time PCR were resolved on agarose gel to confirm the specificity of the PCR. Myoblasts transfected with targeting oligonucleotides revealed amplification of a specific product with the correct predicted molecular size. Direct sequencing of the PCR products demonstrated the presence of the single base pair conversion responsible for restoring the expression of wild-type dystrophin.

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Dystrophin expression in muscle fibers after intramuscular delivery of oligonucleotides Tibialis anterior (TA) muscles of mdx 5cv mice were injected with ssODNs and PNA – ssODNs and dystrophin expression was assessed 2 weeks later as described previously (14). Clusters of dystrophin-positive fibers were detected in all muscles injected with targeting oligonucleotides but not in muscles injected with control oligonucleotides or saline (Fig. 5A). Dystrophin was detected mainly in mature myofibers clustered around the injection site. Occasionally, the distribution of dystrophin appeared patchy, resembling the typical pattern of manifesting carriers of DMD (28). The number of dystrophinpositive fibers was much higher in muscles that received the PNA – ssODNs designed to correct the coding strand (PNA – CORNC) or the non-coding strand (PNA – CORC) of the

mdx 5cv mutation compared with muscles treated with targeting ssODNs made of unmodified bases (Fig. 5B). To confirm that the expression of dystrophin induced by targeting oligonucleotides was not due to an increase in revertant fiber formation, we immunostained cryosections with antibodies against specific regions of the dystrophin protein. In the mdx 5cv strain, a revertant fiber would typically skip exon 10 and probably multiple adjacent exons (14,29), whereas any dystrophin produced as a result of correction of the mdx 5cv mutation would be expected to contain all exons, including exons 10 and 11. In muscles injected with targeting oligonucleotides, all of the clustered dystrophin-positive fibers were stained both by an antibody against more distal regions of the dystrophin protein (exon 31/32) and by an antibody against the exon 10/11 region (Supplementary Material, Fig. 4A), confirming that these are not revertant fibers.


Figure 5. Dystrophin expression in mdx 5cv muscles 2 weeks after injection. (A) Dystrophin expression was localized in mature myofibers clustered around the injection site. Clusters of dystrophin-positive fibers were evident in muscles injected with targeting oligonucleotides but not in muscles injected with control PNA–ssODNs. The number of dystrophin-positive fibers within each individual cluster of fibers expressing dystrophin was higher in muscles that received PNA–CORC and PNA–CORNC when compared with muscles injected with targeting ssODNs. (Scale bar: 100 mm.) (B) Average number of dystrophin-positive fibers per cross-sectional area containing the highest number of positives. Dystrophin-positive fibers were detected throughout the entire length of the muscle. Muscles injected with targeting PNA– ssODNs showed 3–5-fold more dystrophin-positive fibers than ssODNs made of regular bases (n ¼ 5; ∗ P ≤ 0.05, ∗∗ P ≤ 0.005).


Evidence of dystrophin gene correction in injected muscles

Long-term persistence of dystrophin expression in injected muscles Dystrophin was also assessed in TA muscles 4 months following injection of oligonucleotides (Fig. 6). The number of dystrophin-positive fibers at this time point was diminished compared with those detected 2 weeks after injection of oligonucleotides. The number, however, remained higher in muscles injected with the PNA – ssODNs and was comparable between muscles that received PNA – CORC and PNA – CORNC (Fig. 6B). Interestingly, in those muscles, dystrophin was expressed throughout the length of the fiber, suggesting that the loss of dystrophin expression detected was probably related to degeneration of fibers that did not express therapeutic levels of dystrophin (Supplementary Material, Fig. 5). The average length of fibers that expressed dystrophin was significantly higher in muscles that received the PNA– ssODNs targeting the mdx 5cv mutation (Supplementary Material, Fig. 6). Altogether these data demonstrated not only that PNA– ssODNs were more efficient in inducing gene repair, but also that the fibers that were dystrophin-positive at this later time point expressed functional amount of protein to prevent degeneration. These results are consistent with those previously obtained using ectopic expression of dystrophin (30) and suggest that the level of dystrophin expression and its longitudinal distribution along the length of the fiber are clearly important parameters to the long-term functional benefit of oligonucleotide-mediated gene correction strategies for DMD. Western blot analysis was used to confirm expression of dystrophin in muscles 2 weeks and 4 months after ssODN delivery. Dystrophin could be clearly detected in all muscles treated with targeting ssODNs (Supplementary Material, Fig. 7). The level of expression was higher in TA that received PNA– CORC and PNA – CORNC at all time points analyzed, demonstrating that targeting PNA – ssODNs were more

efficient in promoting restoration of dystrophin protein than oligonucleotides made of unmodified bases. No dystrophin was detected in uninjected muscles or muscles that received CTLC and CTLNC. Samples isolated from muscles injected with PNA – CTLC and PNA – CTLNC showed a faint but detectable band of molecular weight identical to that of full-length dystrophin (Supplementary Material, Fig. 7) 2 weeks after ssODN delivery, but not 4 months after delivery, suggesting that PNA – CTLC and PNA – CTLNC had the ability of restoring dystrophin expression through mechanisms independent of gene correction. Characterization of full-length dystrophin transcripts in mdx 5cv muscles To elucidate the effects of ssODNs in vivo, we analyzed TA injected with targeting and control oligonucleotides by realtime PCR. Total mRNA was isolated 2 weeks and 4 months after intramuscular delivery of ssODNs, reverse transcribed and amplified using the Forw-ex9 and the reverse primer Rev-ex10in (Supplementary Material, Fig. 8). No amplification was obtained in samples isolated from uninjected TA muscles or muscles injected with CTLC and CTLNC. Expression of fulllength dystrophin was prominent in muscles that received targeting ssODNs 2 weeks after injection (Supplementary Material, Fig. 8A). Lower but detectable levels of full-length transcripts were also present in muscles that received PNA – CTLC and PNA – CTLNC at this earlier time point. Four months after delivery, the expression had decreased significantly and remained confined only to those muscles that had been injected with targeting ssODNs. Sequencing of all PCR products demonstrated the presence of corrected transcripts in samples injected with targeting ssODNs (Supplementary Material, Fig. 8B and C). Amplicons obtained from tissues injected with PNA – CTLC and PNA – CTLNC 2 weeks after delivery were shown to contain the mdx 5cv mutation (Supplementary Material, Fig. 8B) and were likely to be the result of suppression of the splicing defect at the mRNA level. Gene correction frequencies in vivo We used real-time PCR to estimate the amount of DNA that had undergone gene repair. Genomic DNA was isolated from muscles injected with control and targeting ssODNs, digested with HphI and amplified using the Forw-ex10 primer and the Rev-int10 primer as described above. A product specific to region of the mdx 5cv mutation targeted for repair was detected only in samples treated with targeting ssODNs (Supplementary Material, Fig. 9). The frequencies of gene repair detected 2 weeks after injection were consistently 2 – 3-folds higher in muscles injected with PNA – ssODNs than those obtained in muscles injected with targeting ssODNs made of unmodified bases. The percentage of gene repair ranged between 1 and 2% in muscle that received CORC and CORNC, respectively, as opposed to the 2.8% detected in muscles injected with PNA – CORC and the 3.3% present in muscles that received PNA – CORNC (Fig. 7A). Gene repair was markedly decreased 4 months after delivery, but


To demonstrate further that dystrophin expression in vivo was due to gene correction, we manually excised different regions of cryosections of muscles 3 months after targeting oligonucleotide injections. Total RNA was isolated from those sections, reverse transcribed and subjected to restriction endonuclease digestion using HphI before amplification using the reverse primer complimentary to the 3′ end of exon 10 and the forward primer located in exon 9 of the dystrophin gene. Analysis of the products showed amplification of full-length dystrophin transcripts isolated from regions that contained dystrophin-positive fibers (as determined by immunostaining of adjacent sections) but not from RNA extracted from a region from the same section negative for dystrophin expression (Supplementary Material, Fig. 4B). A separate nested real-time PCR experiment, carried out using the set of primers for the reverse transcription and first amplification reaction followed by a nested PCR, confirmed the results (not shown). Sequencing of the PCR products isolated after fractionation on agarose gels confirmed the T-to-A conversion in all samples analyzed (Supplementary Material, Fig. 4C), thus demonstrating correction at the genomic level.

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remained higher in muscles treated with targeting PNA – ssODNs. Similar results were obtained at the mRNA level (Fig. 7B). Quantitative analysis was performed using mRNA samples isolated from muscles 2 weeks and 4 months after ssODN injection. Samples were reverse transcribed and cDNAs were digested using HphI. Amplification was carried out as described above and monitored by real-time PCR. A product specific to full-length dystrophin and refractory to restriction enzyme digestion were detected only in samples isolated from TA treated with targeting oligonucleotides, but not in samples isolated from TA injected with control ssODNs (Supplementary Material, Fig. 10). Expression of dystrophin was markedly decreased 4 months after injection, but remained

significantly higher in muscles that received the targeting PNA– ssODNs compared with that obtained in muscles treated with ssODNs made of unmodified bases (Fig. 7B).

DISCUSSION We have studied the ability of oligonucleotides made of peptide nucleic acids and containing a single base pair mismatch to correct the genetic defect responsible for the lack of dystrophin in the mdx 5cv mouse. The use of the mdx 5cv model offers several advantages over the more widely studied mdx strain. This mouse model for DMD contains far fewer revertant fibers, a phenomenon that has been attributed


Figure 6. Persistence of dystrophin expression after oligonucleotide injections. Tibialis anterior muscles of mdx 5cv mice were analyzed for expression of dystrophin 4 months after injection of PNA–ssODNs. (A) Dystrophin-positive fibers were evident in all muscles injected with targeting oligonucleotides but not in muscles injected with the control PNA–ssODNs, demonstrating that, in those fibers, gene correction was stable. Expression was confined primarily to small clusters. The intensity of the fluorescence staining in each cluster was comparable with that achieved in sections isolated from wild-type mice. (Scale bar: 50 mm.) (B) The number of dystrophin-positive fibers remained higher in muscles injected with targeting PNA– ssODNs, suggesting that the presence of a direct correlation between frequencies of gene repair and long-term stability of the repair process into muscles. No statistical significance was observed between number of positive fibers in PNA– CORC and PNA–CORNC or between CORC and CORNC injected muscles (n ¼ 4; ∗ P ≤ 0.04, ∗∗ P ≤ 0.005).


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primarily to the location of the mutation (31). Correction in this strain can be demonstrated using restriction analysis and direct sequencing of PCR products obtained after amplification of the region of the dystrophin gene targeted for repair. Furthermore, restoration of full-length dystrophin expression can be quantified at the mRNA level after gene repair allowing for precise determination of frequencies of gene correction mediated by oligonucleotides (14). We have shown that PNA– ssODNs can achieve frequencies of gene correction up to 10-fold higher than those detected using oligonucleotides made of unmodified bases. Correction was demonstrated at the genomic level in both replicating myoblasts maintained in culture for at least 1 week after transfection (Fig. 3) as well as in vivo after direct intramuscular injection (Supplementary Material, Fig. 4). Quantitative

analysis revealed that ssODNs targeting the non-transcribed strand of the dystrophin gene were more efficient than those targeting the transcribed strand by both in vitro and in vivo analyses. These results are in agreement with those obtained by our laboratory, demonstrating that gene correction mediated by oligonucleotides in muscle cells can occur in replicating cells as well as non-dividing cells, suggesting that PNA– ssODNs may share similar mechanisms as ssODNs (14). Our studies clearly indicate that dystrophin expression can be restored using PNA – ssODNs targeting either the transcribed or the non-transcribed strand of the dystrophin gene. No effects were observed in vitro or in vivo, either, using control oligonucleotides made of regular bases perfectly homologous to the coding or the non-coding strand of the dystrophin gene targeted for repair (14). Control PNA – ssODNs


Figure 7. Dystrophin gene repair in mdx 5cv muscles after injection. (A) Gene repair achieved after intramuscular injection of targeting oligonucleotides was estimated using real-time PCR. DNA was isolated 2 weeks and 4 months after delivery of oligonucleotides and digested with HphI. Correction was consistently higher in muscles that received PNA–ssODNs at all time point analyzed (n ¼ 4; ∗ P ≤ 0.03, ∗∗ P ≤ 0.004). (B) Quantitative analysis of full-length dystrophin transcripts confirmed that PNA– ssODNs were more effective in correcting the mdx 5cv mutation than oligonucleotides made of regular bases. Frequencies of repair are expressed as percentage of dystrophin mRNA relative to that of wild-type (C57) muscles. All transcripts were normalized to GAPDH (n ¼ 4; ∗ P ≤ 0.02, ∗∗ P ≤ 0.003).

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repair mechanisms as the frequencies of gene repair detected in replicating cells in cultures were comparable with those in post-mitotic nuclei. Furthermore, our ability to detect gene correction in cells that have been maintained in replicating state over several days and weeks suggests that the repair is stable over time. The beneficial effects achieved by the PNA – ssODNs were evident not only in an increase in the number of dystrophinpositive fibers in a given muscle (Fig. 5), but also in an increase in the distribution of dystrophin within individual fibers. Those increases were shown to beneficially affect the therapeutic efficacy of ssODN-mediated correction of the dystrophin gene over time. The persistence of dystrophin expression for up to 4 months after delivery in a subset of the fibers that were originally targeted for repair demonstrates that the amount of dystrophin protein being restored in those fibers was sufficient to prevent their necrotic degeneration. One of the major challenges in developing efficient gene therapy approaches for muscle diseases is that muscle cells are multinucleated syncytia and the rescue of a single muscle fiber requires correction of multiple nuclei along the length of the fiber. The distribution of the dystrophin protein from a single nucleus is restricted to a limited region of the fiber comprised within its nuclear domain and thus gene correction in a single nucleus may be able to protect only a small region of the fiber. Oligonucleotide-treated muscle fibers that expressed lower levels of dystrophin at early time points remained susceptible to degeneration at later time points. The loss of dystrophin expression detected over time clearly demonstrates that correction of a fraction of nuclei within each myofiber is not sufficient to prevent fiber necrosis. Thus, therapeutic effects in DMD patients require not only oligonucleotides capable of increasing the repair process, but also efficient delivery systems aimed at ensuring correction of a large number of nuclei within the myofiber. The concept of longitudinal distribution of dystrophin and functional activity is not restricted only to the use of gene correction strategies, but has relevance for any gene therapy approach that is currently under development (30). It is difficult to ascertain the minimal number of nuclei that need to be corrected within each individual myofiber to guarantee the persistence of dystrophin expression. The high intensity of dystrophin staining around the periphery of the fiber detected at 4 months after injection and the extensive pattern of dystrophin expression detected along the length of the fiber (Supplementary Material, Fig. 5) suggest that a large number of nuclei had undergone gene repair within fibers that were no longer susceptible to necrosis. That dystrophin staining could be followed in serial sections for up to two-thirds of the total length of the fiber in the TA muscle suggests that the distribution of oligonucleotide is limited in the cross-sectional plane of injection, but that the fibers that do take up the oligonucleotides distribute them widely along their longitudinal axes, allowing for extensive gene correction within the nuclei of that fiber. In this regard, the use of PNA – ssODNs represents a major technological advancement toward the development of effective treatments of muscle disorders using gene correction strategies. Approaches focused at further increasing the level of gene repair are likely to have important implications on the devel-


lacking the specific mismatched base were unable to restore dystrophin expression in muscle cells maintained in culture for 1 week after transfection and then induced to differentiate. Only limited effects were achieved in vivo after direct intramuscular injection of PNA – CTLC and PNA– CTLNC. Restoration of dystrophin expression in those muscles is likely to be due to the ability of PNA – ssODNs to redirect splicing of the dystrophin mRNA as demonstrated by direct sequencing of the full-length dystrophin products obtained by real-time PCR (Supplementary Material, Fig. 8). These results are consistent with those obtained by other groups using different mouse and human cell lines and that have demonstrated that antisense oligonucleotides with strong binding affinity for mRNA like AMO and PT oligomer can correct aberrant splicing at the mRNA level (32– 34). In addition, our studies suggest that oligonucleotides complementary to cryptic splice site can also redirect splicing of the mRNA presumably by binding to the consensus sequences of the U1 snRNP and interfering with the spliceosome machinery during processing of the premature mRNA. Quantitative analysis indicated that full-length dystrophin transcripts present in muscles injected with control PNA – ssODNs were 4 – 5-folds less abundant than those obtained from muscles injected with targeting PNA – ssODNs and 3 – 4-fold those of TA that received targeting oligonucleotides made of regular bases (not shown). Suppression of the splicing defect at the mRNA level mediated by control PNA – ssODNs was only transient as no expression was detected in muscles injected with PNA – CTLC and PNA – CTLNC 4 months after delivery. Dystrophin was clearly detectable in all muscles injected with all targeting ssODNs at all time points analyzed. The differences in gene correction between PNA – ssODNs and unmodified ssODNs were not the result of differences in the uptake of oligonucleotides, as demonstrated by FACS analysis of myoblasts transfected with ssODNs and PNA – ssODNs and analyzed 24 h after transfection (Supplementary Material, Fig. 2C). Rather, the higher gene correction efficiency of PNA – ssODNs probably results from an increased capacity to promote invasion within the double helix of the genomic DNA, thus allowing the DNA to unwind and facilitating homologous pairing of oligonucleotides with their target (35). Differences in fluorescent intensity detected in cells after transfection suggest the presence of different mechanisms of uptake of oligonucleotides made of PNA bases when compared with classic ssODNs (Supplementary Material, Fig. 2A and B). Those differences, however, do not appear to limit the ability of PNA – ssODNs to cross the extracellular membrane of myofibers since no significant differences in oligonucleotide uptake were detected after direct intramuscular injection of ssODNs compared with PNA – ssODNs (data not shown). Taken together, our results demonstrate that the use of oligonucleotides with higher DNA binding affinity can promote the interaction of the ssODN with the genomic DNA targeted for repair greatly reducing the amount of oligonucleotides that has to be introduced into the cell to achieve a therapeutic effect. Previous studies have suggested that replication is an important parameter in achieving higher frequencies of gene repair (26,36 –38). Our data, however, clearly demonstrated that replication is not a limiting factor for the activation of the


MATERIALS AND METHODS Mice Mice of the mdx 5cv (B6Ros.Cg-Dmd mdx-5Cv/J) strain and the control C57 (C57BL/6J) strain were obtained from the

Jackson laboratory and were handled in accordance with guidelines of the Administrative Panel on Laboratory Animal Care of the University of California, Los Angeles. Oligonucleotide synthesis Oligonucleotides CORC and CORNC were purchased from MWG Biotech, Inc. (High Point, NC, USA). The PNA– ssODNs were synthesized by Panagene, Inc. (Panagene, North Korea) or Biosynthesis, Inc. (Lewisville, TX, USA). The targeting oligonucleotides were designed to induce a T-to-A transversion at position +1104 of the mdx 5cv dystrophin gene (Fig. 1). Each control oligonucleotide (PNA–CTLC and PNA–CTLNC) differed from its corresponding targeting oligonucleotide by a single base and was otherwise perfectly homologous to the same region of mdx 5cv dystrophin gene targeted for repair. The ssODNs, CORC, PNA–CORC and PNA–CTLC were complementary to the transcribed strand; CORNC, PNA–CORNC and PNA–CTLNC were complementary to the non-transcribed strand. As additional control, we used ssODNs made of regular bases perfectly homologous to the coding or the non-coding strand of the mdx 5cv mutation, as described previously (14). All oligonucleotides were HPLC purified and exhibited a single peak of the expected molecular weight as determined by MALDI-TOF mass spectroscopy analysis. Cell culture and transfection Cells were derived from limb muscle of neonatal mdx 5cv and C57 mice as described previously (14). For growth, cells were plated on dishes coated with 5 mg/ml laminin (Life Technologies, Inc.) and maintained in growth medium (GM) consisting of Ham’s F10 nutrient mixture (Mediatech, Herndon, VA, USA) supplemented with 20% fetal bovine serum, penicillin and streptomycin. Cell differentiation was induced by maintaining the cells in low serum medium (differentiation medium) consisting of DMEM supplemented with 2% horse serum, penicillin and streptomycin. Myoblasts were plated in wells of 6-well dishes (2 × 104 cells/well) 12 h prior to transfection. Oligonucleotide vectors were resuspended at a concentration of 150 pmol/ml. For each transfection, 5 ml of oligonucleotides were complexed with 5 ml of LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature in a total volume of 0.5 ml of Ham’s F10 nutrient mixture. The complex was then added to the wells containing 1.5 ml of complete GM. Transfection was stopped after 10 h by replacing the transfection solution with fresh GM. Cells subsequently induced to differentiate were maintained in GM for at least 1 week before changing to differentiation medium. Flow cytometry Uptake of oligonucleotides was measured using a Beckton Dickinson FACScalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were trypsinized and harvested at different time after transfection, resuspended in 0.5% BSA, 2 mM EDTA in PBS and processed immediately. For each analysis, a total of 5 × 105 cells were used. Experiments were repeated in


opment of a therapeutic approach of ssODN-mediated gene correction into a clinical scenario. Those include, but are not limited to, the use of different oligonucleotide structures such as morpholino, PS or LNA. Those chemical modifications have proven to be capable of forming stable DNA/ DNA complexes and may be less susceptible to degradation from endonucleases (39– 41). Similar chemical modifications may be applied to ssODNs to further increase the repair efficiency, especially in vivo. Studies aimed at using antisense-mediated technologies to restore dystrophin gene expression by modulating expression of the dystrophin mRNA have gained much attention in the past few years thanks to the promising results obtained in animal models for DMD (5,42– 45) as well as in recent clinical trials in Duchenne boys (3,46). This approach, however, provides only temporary effects and its application into the clinic is limited by the need to deliver continuously the antisense oligonucleotides to the cell nuclei. Gene-editing strategies have clear advantages in diseases where large amounts of tissue needs to be targeted to achieve an effect in terms of both duration and cost of the procedure although multiple administrations of ssODNs may still be required to achieve therapeutic effects. The development of delivery systems to enhance the uptake of PNA – ssODNs into muscles is likely to have important clinical implications for the treatment of DMD. Recent studies have demonstrated that cell penetrating peptides (CPPs) can significantly improve the ability of oligonucleotides to cross the plasma membrane of eukaryotic cells (47– 49) and have opened a new field focused almost exclusively on identifying peptides with membrane-crossing activities (50 –54) that can be tagged to oligonucleotides to promote their delivery in vivo. Great results have already been obtained using antisense technologies. CPP conjugated to morpholino oligonucleotides (CPP – AMOs) have proved to be able to deliver oligonucleotides into mdx muscles and to restore significant amounts of dystrophin expression after intraperitoneal or intravenous administration (45,55– 59). Recent data obtained by several independent groups suggest that CPP conjugated to PNA oligonucleotides can also form stable DNA/ DNA duplexes with a binding affinity higher than that observed for CPP– AMOs (60– 62). The use of CPP– PNA– ssODNs might have profound effects on the overall gene repair frequencies that can be achieved after delivery into skeletal muscles of dystrophin-deficient mice in terms of distribution of dystrophin expression, as well as long-term therapeutic effects achieved after delivery. PNA – ssODNs represents a technological advancement that demonstrates that stable restoration of dystrophin expression can be achieved in muscles with relatively high frequencies of gene repair and their use will have important implications for the development of a treatment to DMD that can be advanced into the clinic.

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triplicate for each time point analyzed and a total of three independent experiments were performed. Intramuscular injections

Western blot analysis Cells were lysed in RIPA buffer (50 mM Tris– HCl, pH 7.4, 150 mM NaCl, 0.5% deoxycholate and 1% Nonidet P-40) containing aprotinin (20 mg/ml), leupeptin (20 mg/ml), phenylmethylsulfonyl fluoride (10 mg/ml) and sodium orthovanadate (1 mM). Total protein in the extract was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Dystrophin immunoblot analysis was performed as previously described (12). Of total protein from mdx 5cv sample, 350 mg was separated by electrophoresis (10 mA for 15 h) using 5% SDS – polyacrylamide gels, and then transferred (250 mA for 7 h) onto nitrocellulose membranes. Membranes were probed overnight at 48C with an antibody directed toward the rod domain [MANDYS -8 (Sigma); 1:400] of the dystrophin protein as described previously (12,14,15). The membranes were blocked with 5% milk in PBS for 1 h at room temperature. Blots were washed with 0.05% Tween-20 in PBS and then incubated with a horseradish peroxidase-coupled anti-mouse secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Specific antibody binding was detected using an enhanced chemiluminescent system (Amersham). Expression of dystrophin protein in vivo was assessed as described previously, with minor modifications (45). Tissue sections were obtained at regular intervals throughout the length of the muscles and grounded into powder. The tissue powder was lysed with 200 ml of RIPA buffer. Immunoblot analysis was performed using 250 mg of total protein. As controls, we used increasing concentrations of protein isolated from 3-month-old C57BL/6J TA muscles (2.5, 5, 7.5, 10 and 12.5 mg) mixed with protein isolated from uninjected mdx 5cv muscles to a final concentration of 250 mg. Dystrophin protein was detected by western blotting as described above using the DYS2 monoclonal antibody (1:200, Vector, Inc., Burlingame, CA, USA). a-Actin was used as a sample loading control (not shown) and was detected using a rabbit antiactin antibody (Sigma). Genomic DNA and restriction endonuclease analysis Gene correction was assessed in vitro using cultured myoblasts. Isolation of DNA for in vivo studies was performed on muscle

Analysis of transcript levels Total RNA was extracted from cultured myotubes or collected sections using TRI-REAGENT (Sigma). For each reaction, 5 mg of RNA was treated with 1 U of DNase I (GIBCO/ BRL) at room temperature and reverse transcribed using the first-strand cDNA synthesis kit (Invitrogen) in the presence of SuperscriptIII (Invitrogen) to a final volume of 20 ml. Reactions were carried out using a forward primer in exon 10 [Forw-ex10: 5′ -GGAAGCTCCCAGAGACAAG-3′ ] paired with a reverse primer complementary to exon 11 [Rev-ex11: 5′ -CCTTGATGAGATGTCAGATC-3′ ]. After an initial step of denaturation at 958C for 5 min, amplification was performed for 40 cycles at 958C for 1 min followed by annealing at 558C for 3 min and extension at 728C for 2 min. Amplification reactions were terminated by an additional extension step at 728C for 10 min and products were fractionated on 12% non-denaturing acrylamide gels. Bands were visualized by incubating the gels on a 1% (W/V) ethidium bromide solution for 15 min at room temperature.


Tibialis anterior (TA) muscles of 12-day-old mdx 5cv mice were injected with 50 ml of targeting or control oligonucleotides resuspended at a concentration of 75 pmol/ml in PBS. Other controls included non-injected muscles or muscles injected with equal volumes of PBS. Mice were sacrificed at 2 weeks, 3 months and 4 months after injection to assess dystrophin gene correction and expression. A total of five animals for each oligonucleotide were used for each time point analyzed. Muscles to be used for histological analyses were dissected and embedded in TissueTek O.C.T. compound (Sakura Finetek USA Inc., Torrance, CA, USA), snap frozen in liquid nitrogen-cooled isopentane and stored at 2808C prior to sectioning.

sections collected from control muscles and muscles injected with ssODNs. Genomic DNA was extracted using the Wizard Genomic DNA extraction kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Total genomic DNA (2 mg) was digested overnight at 378C with 25 U of HphI, purified using Amicon Microconw-PCR Centrifugal Filter Devices (Millipore Corporation, Bedford, MA, USA), and resuspended in 50 ml of H2O. For each amplification reaction, 5 ml of digested genomic DNA was subjected to amplification using the forward primer [Forw-ex10: 5′ -CGAGCATACATTGCGAGCAC-3′ ] specific for the region of exon 10 located 76 bp downstream the intron 9/ exon 10 splice junction, and the reverse primer [Rev-int10: 5′ -CCTCAGAAACTTGTTCTT-3′ ] complementary to a region of intron 10. The ratios of gene correction obtained by targeting PNA – ssODNs to targeting ssODNs were calculated using a standard DDCt method (22DDCt) (63). All data were normalized to GAPDH which was used as control. The primers used were the same as those described previously (19,30), known to amplify a region of the house keeping gene refractory to HphI digestion. As additional internal control for the amplification reaction and to estimate percentages of gene repair of corrected versus non-corrected sequences, we used equal amounts of DNA resuspended in isomolar concentrations of salt and glycerol and were incubated overnight at 378C. Each sample was run in triplicate or quadruplicate in a 20 ml reaction using the ABI SYBRw Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). All reactions were performed on a My iQ single-color detection system (Bio-Rad). After an initial step of denaturation at 958C for 10 min, amplification was performed for 40 cycles at 958C for 1 min followed by annealing at 558C for 2 min and extension and data collection at 728C for 1 min. DNA products were fractionated on 1.5% agarose gels in Tris-acetate/EDTA buffer. PCR products were purified using the Qiagen gel extraction kit (QIAGEN, Inc., Valencia, CA, USA) and DNA sequencing was carried out using an Applied Biosystems ABI377 automated sequencer.


[MANEX1011D (13); 1:50] to confirm the expression of fulllength dystrophin. Specific antibody binding was detected with the Alexa 546-coupled goat-anti-mouse secondary antibody (1:1000). Reduction of the non-specific binding of the secondary antibody was achieved using a papain-digested whole goat-anti-mouse antibody at a concentration of 25 mg/ml as described previously (11,64). Dystrophin-positive fibers were counted for each section and throughout the muscle length. Weakly positive fibers were excluded from the counting. For each time point, a total of five muscles per oligonucleotide injected were analyzed. Statistical analysis Data are presented as means and standard deviations. Comparisons between groups were done using Student’s t-test assuming two-tailed distribution and unequal variances.

SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS The authors would like to thank Dr Glenn Morris (MRIC Biochemistry Group, North East Wales Institute, Wrexham, UK) for kindly providing the MANDYS 1011 antibody. Conflict of Interest statement. None declared.

FUNDING Immunofluorescence analyses Dystrophin immunostaining of cultured cells was performed as described previously (12,14). Briefly, cells cultured on coverslips were fixed in ice-cold ethanol for 10 min, rinsed in PBS and permeabilized in 0.3% Triton X-100 for 10 min at room temperature. A solution containing 5% heat-inactivated normal goat serum and 1 mg/ml BSA in PBS was used as blocking solution for 30 min. Cells were then incubated with primary antibody (MANDYS-8; 1:200) overnight at 48C. Cells were washed in PBS before incubation for 1 h at room temperature with an Alexa 546-coupled goat-anti-mouse (H + L) (Molecular Probes; 1:250) or an FITC-conjugatedgoat-anti-mouse (whole molecule) (Cappel ICN Pharmaceuticals, Inc., Aurora, OH, USA; 1:200) secondary antibody. Coverslips were mounted using Vecta Shield (Vector Laboratories, Inc.) for fluorescence microscopy. Dystrophin staining of muscles was done using serial 10 mm sections (11). TA muscles were cut for at least two-thirds of the muscle length at intervals of 400 mm and examined for dystrophin expression. Air-dried sections were rehydrated in physiologic solutions and blocked using a solution containing normal goat serum diluted 1:20 in PBS. Consecutive sections isolated from injected muscles were immunoassayed using an antibody against the rod domain (exons 31/32) of the dystrophin protein (MANDYS-8; 1:100) or with an antibody specific for the region of the dystrophin protein encoded by exons 10 and 11

This work was supported by grants from the Muscular Dystrophy Association (USA #4331) and the End Duchenne Grant Award from the Parent Project Muscular Dystrophy (PPMD) to C.B.

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Quantitative analysis of full-length dystrophin transcripts was performed using real-time PCR. First-strand cDNA were digested overnight with 5 U HphI in a final volume of 40 ml. PCR reactions were carried out using 4 ml of digested product in the presence of a forward primer homologous to exon 9 [Forw-ex9: 5′ -GTTATGCCTTCACAGGCTGC-3′ ] and a reverse primer (Rev-ex10in: 5-CACTTCTTCAACAT CATTTG-3′ ) in the region of exon 10, spanning the +1131 to +1111 region of the dystrophin transcript that is spliced out in the mdx 5cv strain (Fig. 1). Real-time PCR was performed using a My iQ single-color detection system at the following thermal cycler conditions: 958C for 10 min followed by 40 cycles of a three-step reaction consisting of denaturation at 958C for 30 s, annealing at 558C for 1 min and extension and data collection at 728C for 30 s. The relative expression ratio of full-length dystrophin transcripts obtained in samples treated with PNA – CORC versus CORC and PNA – CORNC versus CORNC was calculated based on real-time PCR efficiencies using a standard DDCt method (63). GAPDH was used as internal standard control for all samples and to normalize transcript levels. Percentages of gene correction in myotube cultures were calculated from a standard curve generated using a dilution series of mRNA isolated from wild-type (C57BL/6J) myotubes mixed with mRNA obtained from untreated mdx 5cv myotubes to a final amount of 2 mg. RNA was reverse transcribed and cDNA was digested with HphI as described above. Amplification was carried out using the Forw-ex9: and the Rev-ex10in and transcripts were normalized to GAPDH. Amplicons were separated on 1.5% agarose gels and PCR products were purified and sequenced as described above.

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