Progress in Gene Therapy Research (Horizons in Cancer Research; Vol. 20)

The ideal aim of gene therapy approach for the treatment of inherited disorders should involve a lasting and tissue specific expression of the functional gene. Besides, in gene therapy any technology used should fulfil several requirements, including safety, simplicity of use, cost effectiveness and amenability to industrial scale. To this end an in situ permanent correction of the defective endogenous gene (gene targeting approach) is preferable to a transient addition of exogenous non-integrating vectors expressing the wild type version of the gene or its cDNA (gene augmentation approach). In fact, the site-specific modification leads to a long term and genetically inheritable expression of the corrected gene, regardless its size. Moreover, in terms of time and space an in situ correction approach allows physiological expression of the targeted gene, since it remains under cell-specific regulatory regions. Gene targeting therapeutic approaches can be broadly divided into viral and non-viral gene transfer technology. Viral vectors take the advantage on the easy integration of the gene of interest into the host and high probability of its long–term expression but they are plagued by safety concerns. although less efficient at introducing and maintaining foreign gene expression Non-viral vectors (naked DNA fragments and plasmid DNA), have the profound advantage of being non-pathogenic and non-immunogenic. 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. Since the viral based approaches are being investigated as well, the current chapter focuses on the use of naked DNA for the production of targeted alterations in the genome of mammalian cells. In particular it will review the most representative experimental strategies recently exploited, trying to consider advantages and down sides of techniques such as the non viral Sleeping Beauty transposon system (SB), the Short Fragment Homologous Recombination (SFHR), the chimeric RNA/DNA Oligonucleotides (RDO), the small interfering RNA (siRNA) and the homologous recombination dependent Gene Targeting (hrdGT).