Progress and Prospects: targeted gene alteration (TGA)

Abstract

Targeted gene repair or targeted gene alteration is a molecular strategy that aims to correct single base mutations responsible for genetic diseases. The concept involves using singlestranded DNA oligonucleotides to direct a nucleotide exchange reaction at the genomic site of the mutation. Investigators have made significant progress in elucidating the mechanism(s) by which the mutation is corrected and have begun to focus on several viable targets that show great potential for clinical application. During the past several years, the field has witnessed a phase transition as the focus has switched from purely basic science to a sustained translational mode. We highlight the important advances over the last two to three years, some of which have moved the technology closer to the clinic while some others have introduced new reasons for caution.

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

“…Although the origin of this variability as well as the mechanisms of action at the molecular level are poorly understood, DNA repair enzymes are involved in this process mainly through the activation of the mismatch repair and/or nucleotide excision repair pathway (Andersen et al, 2002;de Semir and Aran, 2006;Engstrom and Kmiec, 2008;Igoucheva et al, 2004bIgoucheva et al, , 2006Parekh-Olmedo and Kmiec, 2007). The oligonucleotide hybridizes at the targeted location in the genome to create a mismatched base-pair(s) which acts as a triggering signal for the cell's repair enzymes.…”

Sustained Transgene Expression Using Non-Viral Enzymatic Systems for Stable Chromosomal Integration

“…The scope of the present review does not include approaches involving oligonucleotides that were recently reviewed [20,21] but concentrates on approaches involving enzymes such as endonucleases, recombinases and transposases. Their natural properties of integration efficiency and specificity, their genotoxic effects and the problems met to sustain transgene expression are synthesized.…”

DNA Oligonucleotides and Plasmids Perform Equally as Donors for Targeted Gene Conversion

“…Typical DNA donors for nonmurine mammalian somatic cells are plasmids that contain the modification site and flanking sequence from as little as 750 bp up to more than 10 kb (reviewed in Laible and Alonso-Gonzalez 2009). In contrast, chemically synthesized, singlestranded oligonucleotides ranging from 25 to about 100 bp have also been shown to trigger targeted gene alterations (reviewed in Laible et al 2006;Parekh-Olmedo and Kmiec 2007).…”