Current Gene Therapy - Volume 6, Issue 4, 2006

It is feasible to restrict transgene expression to a tissue or region in need of therapy by using promoters that respond to focusable physical stimuli. The most extensively investigated promoters of this type are radiation-inducible promoters and heat shock protein gene promoters that can be activated by directed, transient heat. Temporal regulation of transgenes can be achieved by various two- or three-component gene switches that are triggered by an appropriate small molecule inducer. The most commonly considered gene switches that are reviewed herein are based on small moleculeresponsive transactivators derived from bacterial tetracycline repressor, insect or mammalian steroid receptors, or mammalian FKBP12/FRAP. A new generation of gene switches combines a heat shock protein gene promoter and a small molecule-responsive gene switch and can provide for both spatial and temporal regulation of transgene activity.

Lung transplantation is effective life-saving therapy for the treatment of a variety of end-stage lung diseases. However, the application of lung transplantation is hindered by multiple factors such as the shortage of organ donors, early graft failure and chronic graft dysfunction. These problems are related to various lung injuries before and after transplantation including donor brain-death-related lung injury, ischemia, reperfusion and immune-mediated injuries. Gene transfection presents a potential molecular therapeutic solution to modify the transplanted organ such that it is better able to deal with these obstacles. In fact, in many ways lung transplantation is an ideal situation for gene therapy in that: 1) the targeted injuries are predictable (e.g. IR injury), 2) only transient gene expression is needed in many instances, 3) the immunosuppressive regimen necessary to prevent rejection of the transplanted organ attenuates vector-induced inflammation and the immune response to the vectors or the transgene products, and thus effectively augments and prolongs gene expression; 4) the anatomical structure of the lung enables trans-airway access and local gene delivery - as well as re-transfection. A number of issues need to be considered to develop a strategy of gene delivery in lung transplantation: administration route (intra-airway, trans-vascular, intravenous, intramuscular), timing (donor in-vivo, ex-vivo organ transfection or recipient), vector selection and gene selection. Based on our work and the work of others, over the last decade, we present the state of art of in gene therapy in lung transplantation and exciting future directions in the field.

The use of multiple peptide motifs to provide effective gene delivery holds great promise as an elegant, nonimmunogenic approach to gene therapy. The molecular understanding of cell and viral biology provides a strong foundation on which to pursue this objective. Synthetic peptides containing multiple lysines and/or arginines (occasionally ornithines) provide natural polycations for multivalent electrostatic binding of DNA, and for DNA compaction into particles suitable for gene delivery. These cationic peptides can incorporate additional functional motifs (e.g. for translocating DNA into the nucleus) and they can be linked by disulphide bonds to produce high molecular reducible polycations with superior properties for gene therapy. Many factors influence the size, surface charge and stability of peptide/DNA particles. For in vivo use, uncharged particles resistant to disruption by salt and protein, and targeted to tissue-specific membrane molecules, will be required. Entry into the cell is via one of the endocytic pathways, depending on particle size and (in principle) the target cell surface molecule. Peptide motifs for endocytic escape are based mainly on the anionic fusogenic peptide of influenza virus haemagglutinin and on histidine-rich peptides (where the buffering properties of the imidazole group cause osmotic swelling and probably rupture of endocytic vesicles). Once in the cytosol, translocation of DNA plasmids across the nuclear pore complex into the nucleus is a crucial step, because most target cells for gene therapy are either non-dividing or slowly dividing. Nuclear translocation can be achieved by classical nuclear localising motifs, or more simply by (Lys)16 and other cationic peptides.

As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

The advent of sophisticated experimental tools that can probe the molecular pathology of cancer has revealed a number of genes and gene families that could prove attractive targets for cancer therapy. Thus, gene silencing strategies have been envisioned to treat cancer by targeting the cancer cell's capacity to: (I) resist conventional treatment methods (chemotherapy and radiotherapy), (II) promote angiogenesis, and (III) metastasize and/or to survive microenvironments that normally would promote cell apoptosis/necrosis. The realization of such strategies is limited by the lack of pharmaceutically- viable technologies that enable the safe and effective delivery of gene-targeting agents to neoplastic cells following systemic administration. There are many reasons for this, including an incomplete understanding of how cancer cells respond when genes are silenced. Further the pharmacokinetic and pharmacodynamic attributes of gene therapy products are not well understood. This review will discuss gene therapy strategies that have been developed based on gene inhibition by the use of antisense oligonucleotides, ribozymes and RNA interference (RNAi). In this context, several particularly promising targets will be described, with a focus on strategies that have progressed to the stage where clinical trials have been initiated. The review highlights product development strategies that emphasize non-viral systemic formulations and the potential for delivery systems to become an enabling technology for development of effective gene therapy products.

Transgenic animals are of outstanding relevance for medical sciences, because they can be used to model human diseases and to develop gene therapy strategies. A recent development is lentiviral transgenesis: The generation of transgenic animals by lentiviral transduction of oocytes or early embryos. Lentiviral transgenesis is an efficient method to express transgenes in mice and rats as well as in biomedically relevant livestock. Thus, the applications of this technology range from the generation of disease models to gene pharming for human proteins. An important extension of viral transgenesis is the combination of lentiviral gene transfer with RNA interference. Thereby, expression of specific genes can be silenced and loss-of-function models can be generated. Finally, lentiviral transgenic animals can be used to directly evaluate gene therapy strategies that are based on lentiviral vectors prior to their use in humans.