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Human Molecular Genetics Advance Access originally published online on November 24, 2004
Human Molecular Genetics 2005 14(2):221-233; doi:10.1093/hmg/ddi020
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Human Molecular Genetics, Vol. 14, No. 2 © Oxford University Press 2005; all rights reserved

Strand bias in oligonucleotide-mediated dystrophin gene editing

Carmen Bertoni1, Glenn E. Morris2 and Thomas A. Rando1,3,*

1Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA, 2MRIC Biochemistry Group, North East Wales Institute, Mold Road, Wrexham LL11 2AW, UK and 3GRECC, VA Palo Alto Health Care System, Palo Alto, CA, USA

* To whom correspondence should be addressed at: Department of Neurology and Neurological Sciences, Stanford University Medical Center, Room A343, Stanford, CA 94305-5235, USA. Tel: +1 6508583976; Fax: +1 6508583935; Email: rando{at}stanford.edu

Received August 13, 2004; Revised October 13, 2004; Accepted November 9, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Defects in the dystrophin gene cause the severe degenerative muscle disorder, Duchenne muscular dystrophy (DMD). Among the gene therapy approaches to DMD under investigation, a gene editing approach using oligonucleotide vectors has yielded encouraging results. Here, we extend our studies of gene editing with self-pairing, chimeric RNA/DNA oligonucleotides (RDOs) to the use of oligodeoxynucleotides (ODNs) to correct point mutations in the dystrophin gene. The ODN vectors offer many advantages over the RDO vectors, and we compare the targeting efficiencies in the mdx5cv mouse model of DMD. We found that ODNs targeted to either the transcribed or the non-transcribed strand of the dystrophin gene were capable of inducing gene repair, with efficiencies comparable to that seen with RDO vectors. Oligonucleotide-mediated repair was demonstrated at the genomic, mRNA and protein levels in muscle cells both in vitro and in vivo, and the correction was stable over time. Interestingly, there was a strand bias observed with the ODNs, with more efficient correction of the non-transcribed strand even though the dystrophin gene is not transcribed in proliferating myoblasts. This finding demonstrates that strand bias of ODN-mediated gene repair is likely to be due to the specific sequence of the target gene in addition to any effects of transcription. A better understanding of how the efficiency of gene editing relates to the target sequence will offer the opportunity for rational oligonucleotide design for further development of this elegant approach to gene therapy for DMD and other genetic diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the dystrophin gene that result in a complete absence of the dystrophin protein cause Duchenne muscular dystrophy (DMD) (1Go,2Go). The disease is characterized by progressive muscle wasting and degeneration that results in premature death in the second or third decades of life. Gene delivery approaches for DMD include the use of both viral and non-viral technologies to deliver a normal copy of the dystrophin gene to skeletal and cardiac muscles (3Go). As an alternative to the gene delivery paradigm, we and others have been pursuing gene therapy strategies for DMD that involve either gene editing or modification at the RNA processing or translation steps (4Go). Antisense-based strategies have proven to be effective to restore functional dystrophin proteins in the setting of specific mutations, but the beneficial effects are transient because the vectors used to date are not stably expressed (5Go,6Go). We have explored a gene editing approach for DMD using chimeric RNA/DNA oligonucleotides (RDOs or ‘chimeraplasts’) as vectors to mediate genomic modifications (7Go–9Go). These vectors have been successfully used to induce single base alteration at the genomic level in a variety of cell types, with the efficiency of gene repair depending on the cell type targeted for correction and the target sequence (10Go–15Go). One main advantage of this technology is that the modification is permanent without the use of viral vectors. Furthermore, the corrected gene remains under its endogenous regulatory mechanisms thus assuring appropriate temporal and spatial expression.

We have previously demonstrated the ability of RDOs to act at the genomic level and correct the dystrophin gene mutation in the mdx mouse in vivo in mature myofibers as well as in muscle precursor cells that normally participate in muscle turnover (7Go,8Go). Furthermore, we have demonstrated the feasibility of using RDOs to target the intron/exon consensus splice site sequence of the dystrophin gene upstream of the mdx mutation to restore dystrophin expression by induced exon skipping (9Go). The low efficiency of gene editing has prompted us to test for improved delivery methods, ways to enhance intrinsic cell repair machinery and more efficient vectors. Toward this latter aim, we have begun to test a new generation of gene editing oligonucleotides. Recent studies have shown that oligodeoxynucleotides (ODNs), containing no ribonucleic acid moieties, can induce gene correction to levels comparable to those achieved by RDOs in mammalian cells and in yeast (16Go–21Go). ODNs that have been tested are shorter in length than RDOs and are not self-pairing, as are conventional RDOs. As such, ODNs are designed to anneal to only one strand of the target gene. Whether by RDOs or by ODNs, the conversion of a base on one strand would necessarily be followed by a conversion of the corresponding base on the other strand, perhaps by mismatch repair activities, once the vector has dissociated (22Go). It is by this mechanism that base pair conversion results from the targeting of a single strand.

The use of ODNs may present several advantages for gene editing over RDOs. First, ODNs are less expensive to synthesize and therefore the scale of preparation can be easily expanded for gene therapy applications. Second, the stability of oligonucleotides made only of DNA can be dramatically enhanced by modifications at the 5' and 3' ends (17Go). Finally, it appears that the variability of experimental results is lower with ODNs than with RDOs (19Go).

Interestingly, as ODNs are targeted to only one strand of the target sequence, the efficiency of repair of the transcribed and non-transcribed strands can be directly compared. Initial reports indicated that the rate of gene conversion was higher for ODNs targeting the non-transcribed strand, and this was interpreted as possibly being due to interference of gene correction by the transcription machinery (17Go,18Go). However, this does not appear to be generalizable. Subsequent studies have differed in terms of whether there is a strand bias at all and, if so, which strand is preferentially repaired (23Go–27Go). Most of these studies have examined the issue of strand bias by testing exogenous genes introduced by transfection, by studying prokaryotic genes in eukaryotic cells and by studying episomal rather than integrated plasmids; all have involved genes that are actively transcribed. Our studies offered the unique situation of being able to assess gene repair of an endogenous eukaryotic gene, but one that is not transcribed at the time of oligonucleotide-mediated repair. The dystrophin gene is not transcribed until after the onset of differentiation. Thus, we could specifically examine strand bias in the absence of any contribution from transcription itself by studying dystrophin gene repair in replicating myoblasts.

In this report, we have studied the ability of ODNs targeted to either the transcribed or the non-transcribed strands to correct a point mutation in the dystrophin gene, and we have compared their editing efficiencies to RDOs. For this purpose, we have used the mdx5cv strain which contains a point mutation in exon 10 of the dystrophin gene. This mutation creates a cryptic spice site (Fig. 1) and, as a consequence, the mature mdx5cv transcript has a 54 bp deletion at the 3' end of exon 10 (28Go). This splicing aberrancy results in a transcript that is out-of-frame and a premature stop codon appears 95 bp downstream of the mutation. Thus, as in the mdx mouse, there is a complete absence of dystrophin protein. The correction of the mdx5cv point mutation would be expected to restore the wild-type transcript and full-length dystrophin protein. Using this mouse model, we have found that ODNs are at least as effective as RDOs in mediating gene repair, which represents an advance in terms of vector development for a gene editing approach to DMD. We also found that there is preferential base conversion by the ODN that is targeted to the non-transcribed strand offering insight into the mechanism of oligonucleotide-mediated gene editing.



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  		Figure 1. Oligonucleotide design and targeting of the mdx5cv mutation. (A) The A-to-T mutation in exon 10 of the dystrophin gene is responsible for the creation of an aberrant splice site in the mdx5cv mouse. The exclusion of the last 54 bp of exon 10 leads to a shift in the open reading frame of the dystrophin gene and absence of dystrophin expression in this mouse model for DMD. (B) Linear sequence of targeting RDO (MDX71) and ODNs (MDX72 and MDX73). Upper case letters indicate DNA residues and lower case letters indicate 2'-O-methyl RNA residues. Underlined bases correspond to the single base pair mismatch between the targeting oligonucleotide and the mdx5cv mutation. Control oligonucleotides (MDX81, MDX82, MDX83) are identical to their targeting counterparts (MDX71, MDX72, MDX73) except that the underlined base is complementary to, rather than mismatched with, the corresponding genomic sequence. The secondary structure of the chimeric oligonucleotides is also shown. ODNs contain a CY3 molecule at their 5' ends and four phosphorothioate bases at their 3' ends (indicated as ‘tag’) to increase their stability. BLAST search of the mouse genome database confirmed the lack of homology of the oligonucleotides with genomic sequences other than that specifically targeted in the dystrophin gene.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Oligonucleotide structure and pairing
Each targeting oligonucleotide was designed to induce a T-to-A transversion to correct the mutation of the dystrophin gene in the mdx5cv strain (Fig. 1B). The ODN was designed to be complementary to either the non-transcribed (MDX72) or the transcribed (MDX73) strand of the gene. The RDOs shared a 25 bp homology with the targeted sequence except for a single mismatch between the targeting RDO and the genomic sequence. The targeting and control ODNs were designed to have a 40 bp stretch complementary to the target except, again, for the single mismatchs between the targeting vectors and the genomic sequence. The ODNs contained a fluorescent label modification (CY3) at their 5' ends and four unmatched phosphorothioate DNA bases at their 3' ends (indicated as "tag" in Fig. 1B). Different phosphorothioate bases were tested at the 3' ends of the ODNs but no significant differences were obtained in their efficiency to induce dystrophin gene repair (data not shown).

Dystrophin protein expression in oligonucleotide-treated mdx5cv muscle cells
Western blot analysis of mdx5cv cells treated with targeting oligonucleotides was performed 2 weeks after transfection and 72 h after induction of differentiation (8Go,9Go). Expression of full-length dystrophin was detected in cells treated with targeting but not control oligonucleotides (Fig. 2A). Interestingly, muscle precursor cells transfected with the ODN designed to anneal to the non-transcribed strand of the dystrophin gene had higher levels of dystrophin expression than cells treated with the ODN targeting the transcribed strand. All targeting oligonucleotides resulted in the expression of dystrophin that were unchanged in transfected cells maintained as proliferating myoblasts for weeks before differentiation, demonstrating that the correction was stable and hereditable. The induction of dystrophin expression by targeting but not control oligonucleotides was also demonstrated by immunostaining cells transfected with either ODNs or RDOs and then induced to differentiate (Fig. 2B). The pattern of dystrophin protein staining in myotube cultures was similar to what we have described previously in studies of RDO-induced dystrophin gene repair in vitro (8Go,9Go).



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  		Figure 2. Analysis of dystrophin protein expression in mdx5cv cells treated with oligonucleotides. (A) Myotube cultures transfected with targeting (MDX71, MDX72 and MDX73) or control (MDX81, MDX82 and MDX83) oligonucleotides were induced to differentiate and then analyzed for the expression of dystrophin by western blot analysis and immunohistochemistry. Total protein extracted from untreated cells or cells treated with control oligonucleotides (control RDO MDX81 shown here; identical results were obtain with the other control oligonucleotides) (data not shown) show no detectable expression of dystrophin protein. In contrast, cells transfected with targeting RDO (MDX71) confirmed the ability of the RDOs to restore dystrophin expression in muscle precursor cells in vitro. The ODN targeted to the non-transcribed strand of the dystrophin gene (MDX73) produced levels of dystrophin expression comparable with those achieved by the targeting RDO while the ODN targeting the transcribed strand was less effective in restoring dystrophin expression in those cells. (B) Immunohistochemically, dystrophin expression is evident in mdx5cv cells several weeks after transfection with targeting oligonucleotides and 48 h after differentiation thus demonstrating that the restoration of dystrophin expression is stable over prolonged period of time. No staining was detected in cells treated with control oligonucleotides maintained in culture under similar conditions.

 
Detection of mdx5cv mutation correction by transcript analysis
To confirm that the expression of dystrophin detected by western blotting and immunostaining was indeed due to a correction of the mdx5cv mutation, we analyzed dystrophin transcripts from cells treated with targeting and control oligonucleotides. As wild-type dystrophin transcripts differ by 54 bp from the mdx5cv transcript, we analyzed the sizes of products obtained after RT–PCR amplification and restriction digestion (Fig. 3A). Total mRNA was isolated from cells that had been induced to differentiate after having been maintained in culture for at least 5 days after transfection. Products were fractionated on non-denaturing polyacrylamide gels and bands were visualized by direct exposure of the gels to radiographic film. RNA from untreated cells or cells treated with control oligonucleotides resulted in two bands following restriction digestion of the amplification products (Fig. 3A). One of these (band ‘B2’) corresponded to the region of the mdx5cv transcript lacking the last 54 bp of exon 10 and thus was shorter than the corresponding product (band ‘B1’) from wild-type cells. Cultures of mdx5cv cells treated with targeting oligonucleotides were characterized by the presence not only of band B2 but also of band B1, demonstrating the presence of wild-type transcript and providing direct evidence of correction of the mdx5cv mutation.



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  		Figure 3. Evidence of oligonucleotide-mediated gene conversion at the transcript level. (A) Restoration of full-length dystrophin expression was assessed by RT–PCR in total mRNA isolated from cells transfected with targeting or control oligonucleotides 5 days after transfection and 72 h after induction of differentiation. RNA was reverse transcribed using a reverse primer positioned in exon 12 and was subjected to amplification using a forward primer complementary to exon 9. The amplification products were then subjected to restriction digestion with BsphI. Wild-type (WT) dystrophin transcript from the C57 strain would be predicted to yield two products shown diagrammatically below as ‘A’ and ‘B1’. The shorter transcript from the mdx5cv strain would be predicted to yield the same band ‘A’, but a shorter second product shown as ‘B2’. Lanes from mdx5cv cells transfected with targeting oligonucleotides (MDX71, MDX72 and MDX73) revealed not only the expected products indicative of the mdx5cv transcript (bands ‘A’ and ‘B2’) but also the product (band ‘B1’) corresponding to the wild-type transcript. Cells treated with control oligonucleotides (shown here: MDX81, the control chimeric oligonucleotide; identical results were obtained with other control oligonucleotides, MDX82 and MDX83) (data not shown) revealed only the products indicative of the mdx5cv transcript. These results demonstrate that treatment with the targeting oligonucleotides resulted in the restoration of the correct splicing of exon 10. (B) Quantitative RT–PCR was performed to determine the percentage of cells that had undergone oligonucleotide-mediated gene correction. The reverse primer in the reactions was positioned in the region of the dystrophin transcript absent in the mdx5cv muscle cells. Quantitation of dystrophin gene correction was performed by comparing the amplification products obtained from cells treated with MDX71, MDX72 and MDX73 to those obtained by mixing different amounts of total mRNA isolated from mdx5cv and C57 cells. Normalization was based upon amplification of GAPDH.

 
In order to obtain a quantitative estimate of gene correction by analysis of transcript levels, we designed an assay to quantify the relative levels of wild-type transcripts in the cultures (Fig. 3B). RT–PCR was done using a reverse primer complementary to the last 20 bp of exon 10; this primer results in an amplification product only if wild-type transcript is present. Indeed, no amplification was achieved in untreated cells or cells treated with control oligonucleotides thus demonstrating the specificity of the assay. Quantitation was done by determining the levels of amplification products obtained after transfection of cells with targeting oligonucleotides, and then comparing those levels against a standard curve obtained by altering the ratio of wild-type and mdx5cv RNA from cells maintained in differentiation medium for 72 h. The standard curve revealed a nearly linear relationship between the amount of wild-type transcript present and the level of amplification product over several orders of magnitude (Fig. 3B).

Using this standard curve, we were able to obtain estimates of the gene repair efficiencies of the different oligonucleotides. This analysis revealed that the ODN targeting the non-transcribed strand of the gene was more effective than the ODN targeting the transcribed strand (Fig. 3B). The level of gene repair induced by targeting the non-transcribed strand ranged from 0.2 to 5%, comparable to levels achieved by the RDOs, whereas targeting the transcribed strand resulted in gene correction ranging from ~0.1 to 2%. Thus, the apparent strand bias of the ODNs seen by western analysis of dystrophin protein was confirmed by direct analysis of transcript levels.

Evidence of oligonucleotide-mediated single base substitution at the genomic level
As further evidence that full-length dystrophin protein expression and wild-type dystrophin transcript expression were due to correction of the mdx5cv mutation, we analyzed directly genomic DNA from transfected cells. The A-to-T mutation of the mdx5cv mutation creates an HphI restriction site that is absent in the wild-type gene (Figs 1A and 4A). Total genomic DNA isolated from cells transfected with targeting and control oligonucleotides was subjected to restriction endonuclease digestion using HphI prior to PCR amplification. An amplification product specific for the exon 10/intron 10 region of the dystrophin gene was detected in wild-type C57 cells and in mdx5cv cells treated with targeting oligonucleotides (Fig. 4B). No amplification was detected in untransfected mdx5cv cells or mdx5cv cells transfected with control oligonucleotides. Direct sequencing of the 350 bp amplicons demonstrated that the correction had occurred at the genomic level, converting the mdx5cv mutation back to the wild-type sequence.



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  		Figure 4. Evidence of oligonucleotide-mediated gene conversion at the genomic level. (A) 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 mdx5cv mutation was PCR-amplified using primers flanking the exon 10/intron 10 region. (B) The absence of any amplification products in untreated cells or cells treated with control oligonucleotides demonstrated that the genomic DNA had undergone total digestion. In contrast, in cells treated with targeting oligonucleotides, we obtained a specific PCR product whose molecular weight was identical to that obtained from wild-type cells. (C) Direct sequencing of the amplicon confirmed that the correction had occurred at the genomic level and had reversed the A-to-T mutation of the mdx5cv strain.

 
Uptake of fluorescently labeled oligonucleotides in vivo
Fluorescently labeled oligonucleotides were injected into tibialis anterior (TA) muscles of 12-day-old mdx5cv mice at a concentration of 5 µg/µl (7Go). Mice were sacrificed at different times after injection and comparison of oligonucleotide uptake and stability was performed at 24 h intervals after injection and for up to 7 days. At 24 h after injection, uptake of fluorescent oligonucleotides was detectable in muscle fibers clustered around the injection site (Fig. 5). The number of fluorescently labeled fibers ranged from 200 to 400, consistent with previously obtained results with RDOs using similar injection conditions (7Go). The intensity of fluorescence started to decline by 48–72 h after injection. Clear nuclear staining was detected not only in nuclei in the vicinity of the fiber plasmalemma but also in centrally located nuclei of regenerating fibers (Fig. 5). On the basis of residual fluorescence, the ODNs appeared to be more stable than the RDOs: fluorescence from labeled RDOs was virtually undetectable 4 days after injection, whereas fluorescence from labeled ODNs was still detectable up to 6 days after injection.



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  		Figure 5. Uptake of fluorescently labeled oligonucleotides after intramuscular injection. To compare uptake and stability of oligonucleotides, 5 µl solutions of a RDO (MDX71) or an ODN (MDX72 or MDX73) were injected into TA muscles of 12-day-old mdx5cv mice. Assessment of fluorescently labeled fibers was performed at 24 h intervals, up to 6 days after injection. Clusters of fluorescent fibers along the injection site were evident 24 h after injection of all oligonucleotides. Persistence of fluorescence in myofiber nuclei was evident in muscles injected with an ODN 72 and 96 h after injection (arrows).

 
Dystrophin expression in muscle fibers after oligonucleotide injection in vivo
Muscles were injected with oligonucleotides and dystrophin expression was assessed 2 weeks later (7Go). Clusters of dystrophin-positive fibers were detected in muscles injected with targeting oligonucleotides but not in muscles injected with control oligonucleotides or saline (Fig. 6A). Dystrophin was detected mainly in mature myofibers clustered around the injection site. Occasionally, the distribution of dystrophin appeared patchy (Fig. 6), resembling the typical pattern of manifesting carriers of DMD (29Go). Dystrophin expression was also assessed in TA muscles 3 months following injection of oligonucleotides. The number of dystrophin-positive fibers at this time point was comparable with those obtained at 2 weeks after injection.



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  		Figure 6. Dystrophin expression in mdx5cvmuscles after oligonucleotide injection. (A) Dystrophin expression was seen in TA muscles of mdx5cv mice 2 weeks after injection of ODNs as well as RDOs. The expression was localized in mature myofibers clustered around the injection site. (B) TA muscles of mdx5cv mice were analyzed for expression of dystrophin 3 months after injection of oligonucleotides. Dystrophin expression persisted after a prolonged period of time demonstrating that the gene correction was stable.

 
The number of dystrophin-positive fibers in the individual clusters following single intramuscular injections of targeting oligonucleotides was similar for the RDO and the two ODNs, with an average of about 10 fibers per cluster and a maximum of about 30. This is similar to our previous studies of RDO-mediated dystrophin gene correction in the mdx mouse (7Go), a mouse model in which the targeted base pair mutation is located in exon 23.

In mouse models of DMD, as in the muscles of DMD patients, there is an increasing number of ‘revertant fibers’ with age (30Go). Those fibers are characterized by the expression of shortened, in-frame transcripts due to exon skipping and by the expression, therefore, of shortened dystrophin proteins. In order 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 mdx5cv strain, a revertant fiber would skip exon 10 and probably multiple adjacent exons (30Go), whereas any dystrophin produced as a result of correction of the mdx5cv 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 (exons 31/32) and by an antibody against the exon 10/11 region (Fig. 7), confirming that these are not revertant fibers.



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  		Figure 7. Evidence of oligonucleotide-mediated gene correction of the mdx5cv mutation in vivo. Serial sections of oligonucleotide-injected muscles were stained with antibodies directed against specific regions of the dystrophin protein. Positive staining with the antibody recognizing the exon 10/11 region of the protein confirms that the dystrophin-positive fibers are not revertant fibers.

 
Evidence of dystrophin gene correction at the molecular level in injected muscles
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. From these cryosection fragments, total RNA was isolated and RT–PCR was performed. Analysis of the products fractionated by acrylamide gels showed amplification of full-length dystrophin transcript isolated from regions that contained dystrophin-positive fibers (as determined by immunostaining of adjacent sections) but not from RNA extracted from a region of the same section negative for dystrophin expression (Fig. 8A). A separate nested RT–PCR experiment was carried out using the set of primers for the reverse transcription and first amplification reaction followed by a nested PCR that confirmed the results obtained by direct RT–PCR (data not shown). Sequencing of the PCR products isolated after fractionation on agarose gels confirmed the T-to-A conversion (Fig. 8B), thus demonstrating correction at the genomic level.



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  		Figure 8. Evidence of gene correction in vivo by analysis of dystrophin transcripts. (A) Regions were isolated from cryosections of ODN-injected muscles 3 months after injection. Regions that, in adjacent sections, contained clusters of dystrophin-positive fibers were compared with regions that contained no dystrophin-positive fibers. Lanes corresponding to those two regions are labeled ‘dys+’ and ‘dys–’, respectively. Total mRNA was reverse transcribed and amplified using a forward primer located in exon 9 and a reverse primer flanking the portion of exon 10 spliced out in the mdx5cv dystrophin transcript. Amplification products detectable in mdx5cv muscle injected with targeting oligonucleotides demonstrate the presence of wild-type transcripts. (B) Correction at the genomic level was demonstrated in those fibers by direct sequencing of the RT–PCR products.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have studied the ability of ODNs to restore dystrophin expression in the mdx5cv mouse model of DMD by gene editing, and we have compared them to the gene repair abilities of RDOs. Our studies clearly indicate that dystrophin expression can be restored using ODNs targeting either the transcribed or the non-transcribed strand of the dystrophin gene. Quantitative analysis revealed that ODNs targeted to the non-transcribed strand of the dystrophin gene were more efficient than those targeted to the transcribed strand. The correction was shown to occur at the genomic level by both in vitro and in vivo analyses and the correction was stable over prolonged period of time. The use of the mdx5cv mouse model offers an opportunity to obtain a quantitative estimate of gene repair at DNA, RNA and protein levels and is advantageous over the more widely studied mdx strain because of the much lower levels of revertant fiber formation in the mdx5cv strain (31Go).

There is a possibility that PCR artifacts could arise from persistent oligonucleotides in cells or tissues in which gene editing is analyzed using PCR-based methods to test for single base changes (32Go). We have designed in our studies both genomic and transcript sequences so as to avoid any possibility of such artifacts. First, and perhaps most importantly, all of our PCR primers are designed to have little or no overlap with the genomic region to which the oligonucleotides are complementary. Thus, there is absolutely no possibility that the oligonucleotides themselves could serve as templates for the PCR reactions because there is no sequence complementarity between the primers and the oligonucleotides. Second, the possibility that the oligonucleotides could themselves serve as primers in the PCR reactions is excluded by the size of the amplification products. Any aberrant priming off the one of the oligonucleotides would have resulted in amplification of products differing from the observed (and predicted) products by >50 bp for any of the reactions, a difference that would be unequivocally detected. Finally, other aspects of the experimental systems were designed to eliminate any persistent oligonucleotide as a potential confounding feature, even aside from these specific PCR artifacts. For the in vitro studies, experiments were typically done using cells that had been maintained as proliferating myoblasts in culture for up to many weeks after the transfection, thus reducing the amount of oligonucleotide per cell to a trivial level by simple dilution, in addition to the reduction that takes place by degradation. For assessment of gene correction in vivo by analyzing mRNA, samples were subjected to DNase treatment prior reverse transcription and amplification which should have further eliminated any ODN that could have still been present in the myonuclei 3 months after injection. In summary, we are confident that the possibility that persistent oligonucleotides could account for any of the results presented, including the PCR results, is remote, a conclusion also strongly supported by the corresponding studies of gene correction by detection of the dystrophin protein.

Single injections of targeting ODNs or RDOs into muscles of mdx5cv mice resulted in comparable numbers of dystrophin-positive fibers to those seen with targeting RDOs injected into mdx mouse muscle (7Go). In both cases, a single point mutation is targeted for correction, the former in exon 10 and the latter in exon 23. Assessment of the efficiency of gene correction in vitro in mdx cells was found to be in the range of 2–10% as determined by immunoblot analysis (8Go). In the case of the mdx5cv cells, that efficiency was comparable, falling in the 0.2–5% range as determined by both immunoblot analysis and quantitative RT–PCR (Figs 2 and 3). Differences in the results of gene editing studies have been reported over several orders of magnitude in other systems (22Go,33Go). Such differences may be due to different cellular contexts, target variations in terms of both DNA sequence and chromatin structure and differences in oligonucleotide design, purity and formulation. However, a major reason for differences in reported results is that the data are not directly comparable because some studies report efficacies and others report efficiencies, and the standards for determining such values vary widely. In some cases, efficiencies are calculated as levels of protein expression whereas others determine enzyme activity or even a cellular phenotype. For in vitro studies, the denominator is often either the number of cells or the number of cells transfected. Efficiencies are never calculated on the basis of the number of oligonucleotide molecules per cell or per nucleus, because of the obvious difficulty to determining those numbers, even though such ratios would be more direct reflections of correction efficiencies. Given such differences in how studies of gene repair are reported, direct comparisons of reported results are of limited value. Assessing dystrophin-positive fibers as a measure of efficiency is of course confounded by the lack of correlation between the number of dystrophin-positive fibers and the number of corrected alleles, as a dystrophin-positive fiber may result from the correction of few or many alleles within the multinucleated syncytium. It is also not clear what the minimum number of corrected alleles would be necessary in a myofiber for that cell to be counted as dystrophin-positive. Clearly, caution is warranted when comparing gene editing results in different systems using different methods of analysis.

The use of ODN vectors offers several advantages over RDO vectors. First, ODNs are easier and less expensive to synthesize on a large scale. This is particularly relevant to using such vectors in clinical studies where large amounts of oligonucleotides would be needed for therapeutic application. Second, the modified ODNs are very stable (17Go). The absence of ribonucleic acid moieties eliminates any susceptibility to RNases, and the 5' and 3' modifications provide even more stability (17Go,18Go). We observed an increased stability of ODNs over RDOs based upon the persistence of fluorescence following in vivo injections of fluorescently labeled oligonucleotides.

Several reports have shown that the ability of oligonucleotides to induce single base pair alterations seems to depend on the strand of genomic DNA targeted for correction (17Go,23Go,24Go,34Go). Most studies have found that the non-transcribed strand was preferentially repaired. This has been interpreted as possibly being due to the interference by the transcription machinery of ODN-mediated conversion of the transcribed strand (17Go,20Go,24Go), although this interpretation is confounded by the studies demonstrating preferential targeting of the transcribed strand in some cases (23Go,27Go). Only one study to date has examined this phenomenon in an endogenous gene in a mammalian cell (23Go), and all targeted genes have been transcriptionally active. Our results confirm definitively that transcription, per se, is not the reason for strand bias specifically because the target gene in our studies is transcriptionally silent in proliferating myoblasts. The effect of base conversion can only be detected at the transcript or protein level after the cells have been induced to differentiate, and the results from our studies of cells that have been maintained in a proliferative state for weeks after oligonucleotide treatment demonstrate that the differences in correction of the transcribed and non-transcribed strands occurred when the gene was in a transcriptionally silent state. Although, transcription may have a major influence on the efficiency of oligonucleotide-mediated repair and may strongly skew the efficiency toward one strand or the other, our studies argue that other factors, such as the specific targeted sequences, may be important. The particular characteristics that would render a sequence particularly amenable to targeting, such as GC content, remain to be determined.

One of the unique challenges of gene therapy for muscle diseases is the fact that muscle cells are multinucleated syncytia. Thus, for a cell intrinsic defect, the rescue of a single muscle fiber requires correction of multiple nuclei along the length of the fiber. For the expression of dystrophin, the distribution of the protein from a single nucleus is restricted and thus may be able to protect only a small region of the fiber. This is highlighted by the fact that manifesting female carriers of DMD may show many signs of the disease even though they express dystrophin in a large percentage of myonuclei (29Go). On the basis of the transgenic mouse studies, it has been estimated that expression of dystrophin reaching ~20% of that in control muscle is sufficient to protect the muscle from degenerative changes (35Go). It is difficult at this point to derive any conclusion as to the numbers of nuclei that were corrected per fiber by oligonucleotide-mediated gene editing. However, the intensity of dystrophin staining and the fact that dystrophin is homogenous around the periphery of fibers at the site of injection suggests that there was correction of multiple nuclei in these cells. Furthermore, and perhaps more suggestive, the dystrophin staining could be followed in serial sections for up to two-thirds of the total length of fibers in the TA muscle. At the greatest distances from the injection site, dystrophin staining became more patchy, suggestive of a lower percentage of corrected alleles. Taken together, these findings indicate that the distribution of oligonucleotide is limited in the cross-sectional plane of injection, but that the fibers that do take up the oligonucleotide distribute it widely along their longitudinal axes, allowing for extensive gene correction within nuclei of that fiber.

Recent studies have shown the ability of antisense-mediated technologies to restore dystrophin gene expression by targeting exonic or regulatory sequences to redirect the dystrophin splicing machinery to produce functional although shorter forms of the dystrophin protein (5Go,6Go). This approach, although very promising as a non-viral approach to gene therapy, has as a major limitation the need to deliver continuously the antisense oligonucleotides to the cell nuclei as the effect was shown to decline steadily a few weeks after injections and to be almost undetectable 3 months after injection. The use of RDOs or DNA oligonucleotides to correct mutations at the genomic level offers the advantage of not having to deliver the vector continuously. Indeed our data show that the expression of dystrophin in corrected fibers is persistent for prolonged periods of time (for at least 3 months) with no decline in the number of dystrophin-positive fibers over this time. Furthermore, oligonucleotide-mediated gene repair has the potential to alter splicing of the dystrophin gene, as we have previously shown (9Go), thus expanding the targets to include not only point mutations but also frame-shifting deletions that are the main targets of antisense approaches.

Still, the use of oligonucleotide-mediated gene editing faces several hurdles to realize any promise of therapeutic efficacy. The efficiency remains very low and requires the development of more effective vectors. The use of ODNs containing specific modifications at their 5' ends, at their 3' ends, or of the deoxyoligonucleotides making up the vector may be capable of enhancing specific repair mechanisms present in muscle cells. For instance, methylated bases in the oligonucleotides could be used to preferentially activate specific repair systems in cells (36Go). Such modifications could increase gene correction efficiencies by enhancing endogenous DNA repair activities. The continued development of this technology in terms of vector design and vector delivery will be essential to the eventual use of oligonucleotide-mediated gene editing in the clinical setting for the treatment of DMD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
Mice of the mdx5cv strain and the control C57 strain were obtained from the Jackson Laboratory and were handled in accordance with guidelines of the Administrative Panel on Laboratory Animal Care of Stanford University.

Oligonucleotide synthesis
Oligonucleotides were purchased from MWG Biotech Inc. (High Point, NC, USA). The targeting oligonucleotides (RDO: MDX71, ODNs: MDX72 and MDX73) were designed to induce a T-to-A transversion at position +1104 of the mdx5cv dystrophin gene (Fig. 1). Each control oligonucleotide (RDO: MDX81, ODNs: MDX82 and MDX83) differed from its corresponding targeting oligonucleotide by a single base and was perfectly homologous to the same region of mdx5cv dystrophin gene. For the ODNs, MDX72 and MDX82 were complementary to the transcribed strand, whereas MDX73 and MDX83 were complementary to the non-transcribed strand.

Cell culture and transfection
Cells were derived from limb muscle of neonatal mdx5cv and C57 mice as previously described (37Go). For growth, cells were plated on dishes coated with 5 µg/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 six-well dishes (1x105 cells/well) 12 h prior to transfection. Oligonucleotide vectors (10 µg) were complexed with 1 µl of Lipofectamine 2000 (GIBCO/BRL) 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 by replacing the transfection solution with fresh GM. Cells subsequently induced to differentiate were maintained in GM for at least 24 h after transfection before changing to differentiation medium.

Intramuscular injections
TA muscles of 12-day-old mdx5cv mice were injected 25 µg of targeting or control oligonucleotides in a final volume of 5 µl PBS. Other controls included non-injected muscles or muscles injected with equal volumes of PBS. Mice were sacrificed at intervals of 24 h to test for uptake of fluorescently labeled oligonucleotides and at 2 weeks or 3 months after injection to assess dystrophin gene correction and expression. Muscles to be used for histological analyses were dissected and embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA Inc., Torrance, CA, USA), snap frozen in liquid nitrogen-cooled isopentane and stored at –80°C prior to sectioning.

Western blot analysis
Cells were lysed in RIPA buffer (50 mM Tris–HCl, p. 7.4, 150 mM NaCl, 0.5% deoxycholate and 1% Nonidet P-40) containing aprotinin (20 µg/ml) leupeptin (20 µg/ml), phenylmethylsulfonyl fluoride (10 µg/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 (8Go). From each sample, 350 µg of total protein 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 4°C with an antibody directed toward the rod domain [MANDYS-8 (Sigma); 1 : 400] of the dystrophin protein as previously described (8Go,9Go). 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).

Genomic DNA and restriction endonuclease analysis
Cultured myoblasts were rinsed twice with PBS and 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 µg) was digested overnight at 37°C with 25 U of HphI, purified using Amicon Microcon®-PCR Centrifugal Filter Devices (Millipore Corporation, Bedford, MA, USA), and resuspended in 20 µl of H2O. For each amplification reaction, 5 µl 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 upstream the intron 9/exon 10 splice junction and the reverse primer (Rev-int10: 5'-GAGGGACCAGTTTTCCCCGACAC-3') complementary to a region of intron 10. Each amplification mixture contained 25 pmol of appropriate primer, 10% DMSO, 0.5 U Master Taq DNA polymerase (Takara, Panvera Corp., Madison, WI, USA) and 5 mM of each deoxyribonucleotide triphosphate. After an initial step of denaturation at 95°C for 5 min, amplification was performed for 35 cycles at 95°C for 1 min followed by annealing at 55°C for 3 min and extension at 72°C for 2 min. Amplification reactions were terminated by an additional extension step at 72°C for 10 min. DNA products were fractionated on 1.5% agarose gels in Tris–acetate/EDTA buffer. PCR products were purified using Qiaquick PCR spin columns (Qiagen Inc., Valencia, CA, USA) and DNA sequencing was carried out using an Applied Biosystems ABI377 automated sequencer.

Nested RT–PCR
Total RNA was extracted from cultured myotubes using TRI-REAGENT (Sigma). For each reaction, 5 µg of RNA was treated with 1 U of DNase I (GIBCO/BRL) at room temperature and reverse transcribed using one-step RT–PCR (Qiagen). Reverse transcription and first strand amplification were carried out using an outer reverse primer complementary to exon 12 (Rev-ex12: 5'-CAGGTCCAAAGGGCTCTTCC-3') paired with the forward primer homologous to exon 9 (Forw-ex9: 5'-GTTATGCCTTCACAGGCTGC-3'). PCR reactions were carried out following the manufacturer's recommendations with the addition of 10 mCi 32P-dCTP. Reverse transcription was carried out at 45°C for 30 min followed by a denaturation step at 95°C for 15 min. Amplification of cDNA was performed for 30 cycles at an annealing temperature of 56°C for 1 min and an extension time of 1 min at 72°C. Reactions were terminated by an additional extension cycle of 72°C for 10 min. The radioactive PCR reactions were purified using Amicon Microcon®-PCR Centrifugal Filter Devices (Millipore Corporation) and resuspended in 50 µl of H2O. An aliquot of 10 µl was digested for 1 h at 37°C with BsphI and loaded on an 8% non-denaturing acrylamide gel. Bands were visualized after direct exposure of the gel to X-ray film.

Quantitative analysis of full-length dystrophin transcript was performed using the forward primer in exon 9 previously described (Forw-ex9) and a reverse primer (Rev-ex10in: 5'- CACTTCTTCAACATCATTTG-3') in the region of exon 10, spanning the +1131 to +1111 region of the dystrophin transcript that is spliced out in the mdx5cv strain (Fig. 1).

Nested RT–PCR was carried out using the forward primer Forw-ex9 and a reverse primer (Rev-ex10out: 5'-CTCATGAGCATGAAACTGTTC-3') complementary to the last 21 bp of exon 10. PCR conditions were identical to those previously described and nested PCR was performed on 5 µl of first strand amplification reaction using a forward inner primer (Forw-ex9in: 5'-GGAAGCTCCCAGAGACAAG-3') and the reverse primer Rev-ex10in. Amplification was carried out for 30 cycles of 94°C for 30 s, 55°C for 2 min and 72°C for 2 min. PCR products were excised from agarose gels, purified using a Qiagen Gel extraction kit and sequenced using an automated sequencer.

Immunofluorescence analyses
Dystrophin immunostaining of cultured cells was performed as previously described (8Go). 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 4°C. 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-conjugated-goat-anti-mouse (whole molecule) (Cappel ICN Pharmaceuticals Inc., Aurora, OH; 1 : 200) secondary antibody. Coverslips were mounted using Vecta Shield (Vector Inc., Burlingame, CA, USA) for fluorescence microscopy.

Dystrophin staining of muscles was done using serial 10 µm sections as previously described (7Go). 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 [MANEX1011D (38Go); 1 : 50] to confirm the expression of full-length 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 µg/ml as previously described (7Go,39Go).


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from Duchenne Parent Project/NL and Italia and aktion benni & co e.V., Germany to C.B., and by grants from the Muscular Dystrophy Association (USA) to C.B. and to T.A.R.


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 INTRODUCTION
 RESULTS
 DISCUSSION
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