Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques

The major impact of the human genome sequence is the understanding of disease etiology with deduced therapy. The completion of this project has shifted the interest from the sequencing and identification of genes to the exploration of gene function, signalling the beginning of the post-genomic era. Contrasting with the spectacular progress in the identification of many morbid genes, today therapeutic progress is still lagging behind. The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome. The main strategy has traditionally been focused on the introduction of an expression system designed to express a specific protein, defective in the transfected cell. But the numerous deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. Among these one is represented by gene repair based on homologous recombination (HR). Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function. Homologous recombination is an efficient means for genomic manipulation of prokaryotes, yeast and some lower eukaryotes. By contrast, in higher eukaryotes it is less efficient than in the prokaryotic system, with non-homologous recombination being 10-50 fold higher. However, recent advances in gene targeting and novel strategies have led to the suggestion that gene correction based on HR might be used as clinical therapy for genetic disease. This site-specific gene repair approach could represent an alternative gene therapy strategy in respect to those involving the use of retroviral or lentiviral vectors to introduce therapeutic genes and linked regulatory sequences into random sites within the target cell genome. In fact, gene therapy approaches involving addition of a gene by viral or nonviral vectors often give a short duration of gene expression and are difficult to target to specific populations of cells. The purpose of this paper is to review oligonucleotide-based gene targeting technologies and their applications on modifying the mouse genome.