We have studied the ability of a modified-ssODN (m5CpGGFP), capable of binding MBD4 to direct a site-specific modification at the genomic level and to mediate enhanced gene repair compared with an older generation of correcting ssODN (CORGFP) unable to recruit the specific repair mechanism. Binding was demonstrated in vitro using recombinant MBD4 and was specific only to the ssODN containing the methyl-CpG modification (). Gene correction was consistently more effective when the MBD4-binding ssODN was used as compared with an ssODN containing the 5-methylcytosine, but not in a CpG context or to an ssODN with no 5-methylcytosine modifications (). These data were confirmed at the protein, transcript and genomic levels after treatment of cells with targeting ssODNs.
The differences in gene correction detected do not appear to be due to preferential uptake of ssODNs containing the m5
CpG sequence nor to an increase in their stability after transfection, as suggested by FACS analysis of myoblasts transfected with fluorescently labeled control and targeting ssODNs and followed for up to 1 week after transfection (). Although some toxicity was observed in ssODNs containing m5
C, the increase in cell death was only transient and was not specific to MBD4-binding ssODNs as no significant differences were detected between cells transfected with m5
oligonucleotides. Finally, no differences in cell cycle regulation () or number of cell divisions () were observed in cells treated with m5
as compared with the other ssODNs or to sham transfected cells. Together, these data suggest that the differences in gene correction detected in myoblast cultures were not due to the differences in the ability of MBD4-binding ssODNs to influence cell replication, but were likely due to their ability to increase gene repair. The percentages of GFP-positive cells detected over time after ssODN transfection remained stable in all cultures treated with targeting oligonucleotides, but were significantly higher in cells treated with m5
ssODN. These results clearly demonstrate that gene editing in myoblasts is stable over time, confirming the results previously obtained in our laboratory using gene editing strategies for the dystrophin gene (5
). Furthermore, these data demonstrate that the use of methyl-CpG-modified ssODNs activating the BER through MBD4 binding can significantly enhance gene repair.
Restriction enzyme digestion analysis in combination with real-time PCR demonstrated that the use of ssODNs containing m5CpG and capable of binding MBD4 were able to induce up to 10-fold higher levels of gene correction than older generation oligonucleotides lacking any specific modification. In assessing the frequencies of gene correction, we have taken into consideration several parameters that are likely to yield different estimates of efficiency at the genomic, transcript and protein levels. Such variable include the number of copies of the pGFPmut plasmid integrated at the genomic level, level of expression of the plasmid after G418 selection, the possibility of silencing of plasmid gene expression in vitro and the sensitivities of the FACS and western blot analyses to detect low levels of protein expression. The use of quantitative PCR analysis allowed us to detect correction events occurring at the genomic level independently of possible silencing effects and to estimate frequencies of gene repair per total number of vectors integrated in the genome. The possibility that the results obtained were due to PCR artifacts can be excluded on several grounds. First, each real-time PCR performed had several internal controls, including DNA isolated from untransfected cells and cells transfected with the control ssODN and then subjected to PCR analysis after restriction enzyme digestion. The absence of amplification in those latter samples ensured us that the endonuclease digestion had reached completion. Second, PCR analyses following restriction enzyme digestion were repeated in triplicate experiments using different DNA samples isolated after transfection. Third, each run was loaded on agarose gels to confirm that no amplification product, other then the one expected in samples treated with the targeting ssODNs, would be present in the reaction. Finally, PCR products obtained after quantitative analysis were excised from the gel and sequenced to confirm the presence of the desired single base pair alteration.
MBD4 appears to be a major component of the mechanism of gene correction mediated by methyl-CpG-modified ssODNs. Downregulation of MBD4 alone was sufficient to prevent the restoration of GFP expression after modified ssODN treatment, as demonstrated by analysis at both the protein and the genomic DNA levels (). These observations are supported by studies in MBD4 knockout models. Mice lacking MBD4 expression accumulate C-to-T mutations at CpG sites at a rate of 2- to 3-fold higher than wild-type mice (49
). Other repair mechanisms, although important for maintaining genomic integrity in mammalian cells, do not appear to play an active role in recognizing deamination of 5-methylcytosine at m5
CpG sites, nor can they efficiently compensate for the loss of MBD4 in mice. Therefore, the use of ssODNs containing methyl-CpG modifications and capable of annealing to the genomic sequence creating a mismatched T opposite the G in the methyl-CpG can be used to specifically recruit and activate the MBD4 pathway. The increase in the level of gene correction by ssODNs containing methyl-CpG modifications is likely the result of the efficacy of the repair mechanisms activated to precisely recognize and cleave the mismatched base on the genomic DNA targeted for repair and opposite to the ssODN.
These interpretations were also supported by EMSA assays which demonstrated that the binding of MBD4 occurred only when a G:T mismatch was created by the binding of an ssODN containing the m5CpG modification to the target sequence (). The presence of a single methyl-CpG modification on the ssODN was sufficient to recruit MBD4 to the target sequence. Furthermore, the ability of MBD4 to recognize and bind the targeted sequence only in the presence of the methyl-CpG-modified ssODN annealed to the DNA and creating a G:T mismatch is a clear evidence that initiation of the process requires the ssODN to first anneal to the target sequence within the genomic DNA. No MBD4/DNA complexes were detected when full-length MBD4 protein was added to the reaction mixture containing the methyl-CpG-modified ssODN alone or when MBD4 was added to the reaction containing ssODNs without a methyl-CpG even in the presence of the target sequence ().
For therapeutic applications, the levels of gene correction are likely to require higher efficiencies than those currently being achieved in different systems using ssODNs. The use of methyl-CpG-modified ssODNs is a technical advance that enhances gene repair efficiency as it directs the correction event specifically to the genomic sequence targeted for repair, diminishing the likelihood that the repair mechanism might instead alter the ssODN sequence. Understanding additional steps in the biochemical mechanisms that mediate the repair will also lead to further increases in gene repair efficiencies.
MBD4-binding ssODNs, although more effective than unmodified ssODNs, are sequence specific and can only direct conversion of a thymine into a cytosine. Furthermore, because MBD4 recognizes G:T mismatches only in the context of CpG sites, the sequence targeted by the ssODN requires the presence of a guanine immediately 3′ of the base targeted for repair. This further limits the number of mutations that can be targeted for repair in the context of human diseases. However, it should be noted that ssODN-mediated gene editing can be targeted to both the coding and noncoding strands (3
), thus increasing the possible target sequences. In addition, methyl-CpG ssODNs may be useful in oligonucleotide-mediated exon skipping (21
). This approach takes advantage of the ability of ssODNs to target and disrupt consensus sequences necessary for intron/exon splicing and assembly of mature mRNA. The alteration at the genomic level causes the skipping of one or more exons during the assembly of mRNA transcripts, and ssODNs designed to disrupt specific splicing regulatory elements can have therapeutic applications in cases where shorter, in-frame transcripts allow the production of partially functional proteins (21
). This approach has already being employed in a mouse model of DMD using older generations of oligonucleotides with encouraging results (21
). Although intron/exon are not specifically enriched in TG dinucleotides, that are the specific targets of methyl-CpG-modified ssODNs, such dinucleotides are frequently present in consensus sequences and other splicing control elements and are thus potential targets for altering splicing to restore functional protein expression from mutant genes. The ability of methyl-CpG-modified ssODNs to induce single base pair substitutions at the intron/exon boundaries of the dystrophin gene is currently being tested in our laboratory. The development of a successful approach using methyl-CpG-modified ssODNs in oligonucleotide-mediated exon skipping of the dystrophin gene would have a wide range of clinical applications. Its use could be applied to the majority of DMD patients (60–70%) in which large deletions or frameshift mutations could be corrected by restoring the dystrophin reading frame.
Random integration, mispairing and activation of homologous recombination at sites different from those targeted for repair might ultimately preclude this technology from entering a clinical scenario. For approaches aimed at repairing postmitotic muscles cells, those issues remain less of a concern since the effects are likely to be confined within a limited number of cells that have undergone repair. For those approaches aimed at targeting and correct muscle stem cells, a detailed analysis of cells undergone repair will be critical to demonstrate its safety and long-term effects. Those studies will have to focus on individual cells not just a pool of corrected cells so that the effects of the correction process can be determined in detail on a single cell base.
Future studies in our laboratory will be aimed at determining the safety of gene correction process in muscle cells and will be focused primarily on assessing the fate of cells that have undergone repair. The optimization of culturing conditions capable of maintaining in culture clones isolated after cell sorting will be critical to characterize those cells and study changes in gene expression profile in each individual cell that have undergone gene repair. To date, those studies have been problematic due to the inability to expand primary myoblasts isolated using sorting techniques. New technologies are now being developed that will allow us to study expression profiles from single cells and might enable us not only to study the effects of gene repair within each individual cell, but also to determine the effects of ssODNs immediately after transfection. Those technologies might ultimately hold the key in determining the applicability of ssODN-mediated gene correction for the treatment of many genetic defects.
Ultimately, safe and effective treatments of genetic diseases using ssODNs will necessitate synergistic efforts aimed not only at increasing the level of gene repair but also methods to insure efficient delivery of ssODNs to all the cells whose correction would be necessary for maximum therapeutic effect. For diseases in which the tissue being targeted undergoes continuous turnover either under normal physiological conditions or in the setting of the disease, gene correction of the stem or progenitor cells that are responsible for forming new tissue would be essential to insure sustained therapeutic effects. These delivery issues are faced by every form of gene therapy. However, especially for nonviral forms of gene therapy, the efficacy of the vector once delivered to the cell, both in terms of magnitude and duration, will be a critical determinant of the success of that therapeutic approach. The use of methyl-CpG-modified ssODNs is just such a method to enhance the efficacy of ssODN-mediated gene editing, and may have applications across a wide range of genetic disorders.