Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

Abstract

Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.

Genome-wide Methylation Profiles Reveal Quantitative Views of Human Aging Rates

“…This meQTL thus captures a cis -relationship in which rs140692 influences the methylation state of MBD4. That MBD4 plays a role in human aging is supported by previous work linking MBD4 to DNA repair, as well as work showing that mutations and knock-downs of MBD4 lead to increased genomic instability (Bellacosa et al, 1999; Bertoni et al, 2009). …”

Identification of bidirectional gene conversion between SMN1 and SMN2 by simultaneous analysis of SMN dosage and hybrid genes in a Chinese population

“…Of note, a recent in vitro study highlights the significance of our SMN2-to-SMN1 conversion finding by showing that it can even be driven by using single-stranded oligonucleotides [23]. Given that SMN1 genes can produce more stable and biologically active SMN protein, creating an SMN1-like gene converted from SMN2 in SMA patients might be an appealing target for gene therapy [24]. Thus, our finding not only provides evidence of natural conversion events of SMN2-to-SMN1 but also could be the basis of a potential therapy for SMA in the future.…”

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…Support for such a model was recently obtained in murine myoblasts. 18 In alternative models (Figure 1), the ssODN becomes physically integrated into the genome within the context of either a replication fork (Model II) or a displacement loop (Model III).…”