Oligonucleotide-Directed Mutagenesis and Targeted Gene Correction: A Mechanistic Point of View

Abstract

Within the last decade, a number of nucleic acid-based gene targeting strategies have been developed with the ultimate goal to cure human genetic disorders caused by mutations. Thus far, site-directed gene targeting is the only procedure that can make predefined alterations in the genome. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. In addition, targeted specific mutations can be made in the genome for functional analysis of proteins. Several approaches, including chimeric RNA-DNA oligonucleotides, short single-stranded oligonucleotides, small fragment homologous replacements, and triple-helix-forming oligonucleotides have been used for targeted modification of the genome. Due to the absence of standardized assays and mechanistic studies in the early developmental stages of oligonucleotide-directed gene alteration, it has been difficult to explain the large variations and discrepancies reported. Here, we evaluate the progress in the field, summarize the achievements in understanding the molecular mechanism, and outline the perspective for the future development. This review will emphasize the importance of reliable, sensitive and standardized assays to measure frequencies of gene repair and the use of these assays in mechanistic studies. Such studies have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The conventionally accepted but unproven dogmas of the mechanism of gene repair are challenged and alternative points of view are presented. Another important focus of this review is the development of general selection procedures that are required for practical application of this technology.

Commentary: Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

“…OMM makes use of different types of oligonucleotides: single-stranded DNA oligonucleotides containing 5 and/or 3 modified ends to protect the molecule against cellular nuclease activities (Campbell et al, 1989), chimeric RNA/DNA or DNA/DNA molecules D. Breyer et al (Igoucheva et al, 2004a;Parekh-Olmedo et al, 2005), RNA oligonucleotides (Storici, 2008), and triplexforming oligonucleotides (Simon et al, 2008).…”

Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells

“…Previously, we demonstrated that relatively small single-stranded ODNs can cause a sequence-specific, homology length-dependent and strand-specific gene correction of mammalian chromosomal DNA. 31,35 Several assay systems have been established in which phenotypic changes can be detected upon gene correction. All of the developed systems have provided clear evidences of gene correction by ODN in the episome and chromosome of many mammalian cells, including CHO-K1, 31 primary human keratinocytes, 38 melanocytes 37 and mouse ES cells.…”

“…35 In addition, structural modifications, such as shift of mismatch-forming nucleotide from a central position toward to the 3 0 -and 5 0 -ends of ODN had a more negative effect on correction activity of ODN. In fact, the mismatch-forming nucleotide close to the 5 0 -end was much more disruptive than its 3 0 -counterpart suggesting that the initial pairing of the 5 0 -region of ODN with its complementary target might be a crucial step during the strand invasion process.…”

“…In fact, the mismatch-forming nucleotide close to the 5 0 -end was much more disruptive than its 3 0 -counterpart suggesting that the initial pairing of the 5 0 -region of ODN with its complementary target might be a crucial step during the strand invasion process. 34,35 It is interesting to note that not all corrected cells were able to survive and in fact, the only one-third of EGFP + sorted cell produced viable clones. Recently, Olsen et al 49 found that the majority of corrected CHO-K1 cells were arrested at G 2 /M stage and only 2% of the initially EGFPcorrected cells made viable colonies.…”

“…The primary advantage of such an approach is the ability to make a permanent specific alteration into a gene of interest in a single step, without disturbing the genomic organization required for endogenous expression and regulation of the targeted gene. In the past 8 years, we have made significant progress in our understanding of the mechanism responsible for gene alteration by ODNs [32][33][34][35][36] and provided clear evidence that this approach can be used to introduce permanent site-specific changes into episomal and chromosomal DNA of somatic cells 31,37,38 and embryonic stem (ES) cells. 39,40 The goal of the present study was to assess the use of ODN-directed gene repair in MSCs.…”