Editorial [Hot Topic: Recombinase Technology for Gene Therapy (Guest Editor: Zoltan Ivics)]

The ability to efficiently deliver foreign genes into cells offers opportunities to use gene therapy to correct genetic diseases. Effective gene therapy requires robust delivery of the desired genes into the relevant target cells, long-term gene expression and minimal risks of secondary effects. DNA recombinases, enzymes that catalyze recombination between DNA molecules, can be harnessed for stable genomic insertion of therapeutic gene constructs. This Hot Topic Issue of Current Gene Therapy is dedicated to technologies based on recombinases and their potentials for future clinical appications. Inserting genetic information at predetermined locations into the human genome has been the goal of gene transfer technology for almost two decades. Recchia and Mavilio provide an account of site-specific gene insertion by the replicase/integrase protein (Rep) of the adeno-associated virus (AAV), and technologies that exploit this insertion machinery to insert genes at the natural AAV integration site on human chromosome 19. Another promising technology for site-directed gene insertion is based on the φC31 integrase system that mediates the integration of plasmid DNA into the chromosomes of mammalian cells in a sequence-specific manner. Chavez and Calos review how φC31 integrase mediates transgene integration into the genomes of target cells, and summarize the recent preclinical applications of the system to gene therapy. DNA transposons are discrete pieces of DNA with the ability to change their positions within the genome via a ‘cut and paste’ mechanism called transposition. Belay et al. decribe the use of the Sleeping Beauty and piggyBac transposon systems for gene transfer into adult and pluripotent stem cells. In particular, the use of a hyperactive variant of Sleeping Beauty, called SB100X, yields robust gene transfer efficiencies into human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), and induced pluripotent stem cells (iPSCs). These cells are relevant targets for stem cell biology and for regenerative medicine and gene- and cell-based therapies of complex genetic diseases. In the context of iPSC technology, the ability to coax the differentiation of pluripotent stem cells into clinically relevant, transplantable cell types is a key step towards their ultimate use in clinical applications. A major hurdle in ex vivo delivery of non-viral vector systems into relevant primary cell types is the toxicity of the transfection/electroporation protocols that is typically observed. The development of hybrid vector systems combining the natural ability of viruses to traverse cell mebranes with efficient genomic insertion mediated by (otherwise) non-viral DNA recombinase systems is a promising strategy. Staunstrup and Mikkelsen review technologies based on packaging non-viral integration machineries (including zinc finger nucleases, homing endonucleases, the FLP recombinase system and the Sleeping Beauty transposon) into integrase-defective lentiviral particles. The latter combination showed efficient gene transfer in a range of human cell types and an insertion profile favorable to conventional lentiviral vectors. In addition, herpes simplex virus vectors with a tropism to infect neural progenitor cells have been used to target Sleeping Beauty transposon insertions in the central nervous system (CNS) in an in utero gene delivery system in the mouse. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. de Silva and Bowers provide a comprehensive review of the characteristics of the HSV/Sleeping Beauty vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies. Finally, Hausl et al. describe hybrid adenovirus/Sleeping Beauty vectors that have been used to efficiently deliver the transposon vectors expressing the blood clotting Factor IX into the liver in a hemophilic dog model, thereby opening up new avenues for in vivo genome engineering in large animal models. In in vivo gene delivery, the therapeutic gene vector is directly introduced into an organ, where expression of the therapeutic gene construct is desired. The use of physical delivery systems, such as hydrodynamic injection, can overcome the low efficiency of cellular uptake of non-viral gene delivery systems, such as Sleeping Beauty. Hackett et al. discuss the challenges associated with scaleup of this procedure from mice to large animal models. In certain applications, transient delivery of gene products (RNA or protein) might be advantageous over permanent or long term expression of biologically active molecules, including potentially toxic gene products, such as DNA recombinases. Schott et al. review the current state of approaches for the delivery of mRNA and proteins by a variety of viral and cell-based approaches with a focus on RNA viruses, virus-like particles including retrovirus-particle mediated transfer of mRNA or proteins, extracellular vesicles, and cell-penetrating peptides. The reader finds in this issue of Current Gene Therapy selected examples of technologies based on DNA recombinases for gene insertion and genetic engineering in the context of gene therapy. Continued refining and further development of these technologies, as well as a careful assessment of the potential risks associated with their application, will support translation of these DNA recombination systems as well as procedures for their delivery into clinical practice.