Targeted Gene Repair: The Ups and Downs of a Promising Gene Therapy Approach

Abstract

As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

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

“…It is referenced in the literature under other names such as targeted nucleotide exchange, chimeraplasty, oligonucleotidemediated gene editing, chimeric oligonucleotidedependent mismatch repair, oligonucleotide-mediated gene repair, triplex-forming oligonucleotides induced recombination, oligodeoxynucleotide-directed gene modification, therapeutic nucleic acid repair approach, targeted gene repair (see e.g. Andersen et al, 2002;Christensen et al, 2006;Cole-Strauss et al, 1999;de Semir and Aran, 2006;Igoucheva et al, 2006;Zhang et al, 1998).…”

“…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.…”

“…Last but not least, the technique seems to offer opportunities for the future in the field of human gene therapy to correct point mutations, for instance in monogenic inherited diseases and cancer (Christensen et al, 2006;de Semir and Aran, 2006;Kmiec, 2003;Wu et al, 2001). The therapy using oligonucleotides or RNA/DNA chimeras can result in a fraction of cells in which the wild-type gene can be restored.…”

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

“…Such molecules can be exploited to promote homologous recombination or other sequence directed repair between a specific targeted gene segment and an externally supplied gene correction DNA donor or the insertion of externally supplied genetic material at a specified target site in the genome. 1,2 Two of the more promising gene targeting molecules are designed zinc-finger nucleases (ZFNs) and triplex-forming oligonucleotides (TFOs). Indeed using gene targeted ZFNs levels of targeted genetic editing of close to 40% have recently been obtained under optimized conditions in specific cell types.…”

Targeted sequence alteration of a chromosomal locus in mouse liver

“…For example, the dystrophin gene in Duchenne muscular dystrophy patients and the cystic fibrosis transmembrane conductance regulator gene in cystic fibrosis patients are potential targets of this technology (Gruenert et al, 2003). Targeted sequence conversion using oligonucleotides has been developed for introducing small sequence alterations including deletions, insertions, and base-substitutions into mammalian genomic DNA (Andersen et al, 2002;de Semir and Aran, 2006;Igoucheva et al, 2004;Parekh-Olmedo and Kmiec, 2007;Richardson et al, 2002;Seidman and Glazer, 2003). Small double-stranded DNA fragments have also been used to modify genomic DNA in both human and mouse cells (Gruenert et al, 2003;Sangiuolo, F. and Novelli, 2004).…”