Oligonucleotide-based gene targeting technologies

The major impact of the human genome sequence is the understanding of disease aetiology 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 postgenomic era. The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome. It should involve a lasting and tissue specific expression of the functional gene. Besides, in gene therapy preferably any technology used should fulfil several requirements including safety, simplicity of use, cost effectiveness and amenability to industrial scale. The oligonucleotide-base gene targeting strategies now in use are based on the naturally occurred mechanism of homologous recombination (HR). Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function. HR in eukaryotes is less efficient than in prokaryotic system. Anyway 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 and used in the cellular genetic therapy approach. Cellular genetic therapy is the ultimate frontier for those pathologies that are the consequent to a specific non-functional cellular type, by the replacement of sick cells with healthy ones, obtained from the same patient or a different donor and previously cured with gene therapy techniques. The purpose of this paper is to review the oligonucleotides based gene targeting technologies: the small fragment homologous recombination (SFHR), the chimeric RNA/DNA oligonucleotides (RDO), triplex forming oligonucleotide (TFO) and the homologous recombination dependent gene targeting (hrdGT). With reference to the naked DNA approach, the recent;availability of better delivery methods (e.g. electroporation and microinjection) has made the non-viral gene transfer an increasingly more important and viable method for gene therapy.