Current Gene Therapy - Volume 11, Issue 5, 2011

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.

The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. 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. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNStargeted gene therapeutic strategies.

Sleeping Beauty (SB) transposons have been effective in delivering therapeutic genes to treat certain diseases in mice. Hydrodynamic gene delivery of integrating transposons to 5-20% of the hepatocytes in a mouse results in persistent elevated expression of the therapeutic polypeptides that can be secreted into the blood for activity throughout the animal. An alternative route of delivery is ex vivo transformation with SB transposons of hematopoietic cells, which then can be reintroduced into the animal for treatment of cancer. We discuss issues associated with the scale-up of hydrodynamic delivery to the liver of larger animals as well as ex vivo delivery. Based on our and others' experience with inefficient delivery to larger animals, we hypothesize that impulse, rather than pressure, is a critical determinant of the effectiveness of hydrodynamic delivery. Accordingly, we propose some alterations in delivery strategies that may yield efficacious levels of gene delivery in dogs and swine that will be applicable to humans. To ready hydrodynamic delivery for human application we address a second issue facing transposons used for gene delivery regarding their potential to “re-hop” from one site to another and thereby destabilize the genome. The ability to correct genetic diseases through the infusion of DNA plasmids remains an appealing goal.

Gene vehicles derived from lentiviruses have become highly esteemed tools for gene transfer and genomic insertion in a wealth of cell types both in vivo and ex vivo. However, accumulating evidence of preferred insertion into actively transcribed genes, driven by biological properties of the parental human immunodeficiency virus type 1, has questioned the safety of this vector technology. As a consequence, integrase-defective lentiviral vectors [IDLVs], carrying an inactive integrase protein, have been developed and used with success for persistent in vivo gene transfer to quiescent or slowly dividing cells. We and others have shown that episomal DNA delivered by IDLVs may serve as a substrate for heterologous integration machineries, including recombinases and transposases, and homologous recombination triggered by nuclease-induced DNA damage. New vector systems that combine the best of lentiviral gene delivery and nonviral integration systems are under development. The first prototypes of such hybrid lentiviral vectors facilitate efficient gene transfer and show profiles of insertion that are not dictated by the biological constraints of the normal integration pathway and are, therefore, significantly different from the profile of conventional lentiviral vectors. The stage is set for further exploration of these vectors. In this review, we summarize the background and short history of hybrid IDLV-based vector systems and discuss their applicability in gene therapy and treatment of genetic disease.

The Sleeping Beauty (SB) transposase system for somatic integration offers great potential for in vivo gene therapeutic applications and genome engineering. Until recently, however, efficacy of SB transposase as a gene transfer vector especially in large animals was lacking. Herein, we report about the newest viral vector development for delivery of the SB transposase system into large mammals. Over the past decade various hyperactive versions of SB transposase and advanced adenovirus vectors enabling efficient and safe delivery of transgenes in vivo were developed. Already several years ago it was demonstrated that adenovirus vectors can be used for delivery of the SB transposase system into murine liver. Our newest study showed for the first time that a hyperactive transposase system delivered by high-capacity adenoviral vectors can result in somatic integration of exogenous DNA in canine liver, facilitating stabilized transgene expression and phenotypic correction for up to three years in a canine model of human disease. In this review we discuss safety issues and further improvements of this adenovirus based hybrid vector system for somatic integration. In the future this approach paves new paths towards the possible cure of human genetic diseases and novel strategies for in vivo genome engineering in large mammals.

The potential use of the φC31 integrase system in gene therapy opens up the possibilities of new treatments for old diseases. φC31 integrase mediates the integration of plasmid DNA into the chromsomes of mammalian cells in a sequence- specific manner, resulting in robust, long-term transgene expression. In this article, we 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. These applications encompass in vivo studies in liver and lung, as well as increasing ex vivo uses of the system, including in neural and muscle stem cells, in cord-lining epithelial cells, and for the production of induced pluripotent stem cells. The safety of the φC31 integrase system for gene therapy is evaluated, and its ability to provide treatments for hemophilia is discussed. We conclude that gene therapy strategies utilizing φC31 integrase offer great promise for the development of treatments in the future.

The transient delivery of gene products (RNA or proteins) is not a biotechnological invention but rather an evolutionarily conserved process underlying and regulating a variety of biological functions. On the basis of insights into the underlying mechanisms, several viral and cell-based approaches have been developed for the delivery of RNA or proteins. Prominent applications include the induction of major biological or therapeutic effects on the basis of “hit-and-run” mechanisms, such as vaccination, cell fate modification (reprogramming, differentiation), control of cell trafficking, enhancement of cell regeneration, and genome engineering using sequence-specific recombinases or nucleases. Ideally, procedures for delivery of RNA or proteins should be targeted to specific cells, overcome biophysical hurdles without harming cellular integrity, circumvent the various alarm signals of the innate immune system, allow dose-controlled delivery of functional biomacromolecules, and avoid the induction of an adaptive immune response. Here we review the current state of approaches for the delivery of mRNA and proteins 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 basic concepts and recent advances are put into perspective in the context of potential limitations of the technologies and strategies to overcome cellular barriers and defense mechanisms.

Inserting genetic information at precise locations into the human genome has been the goal of gene transfer technology for almost two decades. The spectacular progress of mammalian genetics has led to the development of technology for genome editing and homologous recombination in human somatic cells that is finally approaching efficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locations by enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adenoassociated virus (AAV), to insert genes at the natural AAV integration site on human chromosome 19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis.

Transposon technology is a particularly attractive non-viral gene delivery paradigm that allows for efficient genomic integration into a variety of different cell types. In particular, transposon-mediated gene transfer is a promising tool for stem cell research, by virtue of its ability to efficiently and stably transfer genes into adult and induced pluripotent stem (iPS) cells. Moreover, transposons open up new perspectives for non-viral-mediated stem cell-based gene therapy. Several transposon systems, especially the Sleeping Beauty (SB), the piggyBac (PB) and Tol2, have been optimized for gene transfer into mammalian cells. In particular, SB resulted in stable gene transfer into various adult stem cells including human CD34+ hematopoietic stem cells (HSCs), myoblasts and mesenchymal stem cells (MSCs). This has been confirmed with PB, yielding stable gene transfer in human CD34+ HSCs. Recently, PB transposons were used to deliver the genes encoding the reprogramming factors into somatic cells making it an attractive technology for generating iPS cells. Subsequent de novo expression of the PB transposase resulted in traceless excision of the reprogramming cassette. This prevented inadvertent re-expression of the reprogramming factors obviating some of the concerns associated with the use of integrating vectors. Transposons have also been used as a novel non-viral paradigm to coax differentiation of iPS cells into their desired target cells by forced expression of specific differentiation factors. This review focuses on the emerging potential of transposons for gene transfer into stem cells and its implications for gene therapy and regenerative medicine.

Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and delivery strategies to achieve positive clinical results. The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a cancer vaccine. It has been theorized that effective cancer vaccines can be developed using the minimum essential subset of T cell and B cell epitopes that comprise the ‘immunome’, the universe of neoplasm-derived peptides that interface with B and T cells of the host immune system. Idiotypic antigenic determinants of a B-cell lymphoma lie within the hypervariable regions and mainly within the complementarity-determining regions (CDR)s 3. Thus, the CDR3s are considered a “hot spot” of particular interest for construction of subunit vaccines. DNA vaccines, whose safety and tolerability are substantiated in completed and ongoing clinical trials, have emerged as a novel lymphoma vaccine formulation for antigen-specific immunotherapy. The molecular precision tools offered by gene-based vaccines allow to explore the use of CDR3 sequence as an antilymphoma vaccine.

Gene therapy is an emerging technique with widespread applications in treatment of cardiovascular diseases, monogenic disorder, infectious diseases, and especially cancers. The major challenge for gene therapy is to deliver therapeutic genes to target tissues. Although various gene delivery vectors such as harmless viruses and micro/nano-particles have been developed (i.e. commonly system delivery), concerns remain for the transfection efficiency and stability of those working copies in these vectors. Local gene delivery such as intratumoral infusion, electroporation and implants offers significantly enhanced transfection efficiency with decreased toxicity compared to system delivery and has been broadly used in clinics. In this paper, we reviewed the local gene delivery methods and discussed their distinctive advantages and potential challenges in cancer treatment.