Current Gene Therapy - Volume 6, Issue 6, 2006

Morbidity and mortality attributed to diseases of the heart and blood vessels are growing steadily around the globe, as a result of changing lifestyles and increased longevity. Even in affluent countries many patients with cardiovascular syndromes do not benefit from current conventional therapies. Failure of drugs, catheter-based, and surgical interventions leaves millions of patients in a great need for new therapies. Cell and gene therapy hold great promise for cardiovascular therapies. As most cardiovascular pathologies are confined to a specific organ and are associated with arterial occlusion or muscle damage, both may be amenable to local cell and gene therapies. Local delivery of genes or cells that can promote the formation of new blood vessels (angiogenesis) or lead to tissue regeneration should be achievable with current knowledge and molecular technologies. So far, however, the promise of gene and cell therapy has not fulfilled the expectations. Nevertheless, careful review of the work and of current thinking in the field leaves room for optimism that gene and cell therapy may reach the clinical setting in the near future, providing a new option for many patients. In this paper, we review clinical studies of gene and cell therapy and discuss their impact on future research.

Ex vivo gene therapy is a possible treatment for several muscular dystrophies. The best transgene to be expressed and the appropriate cell type to be used currently remain the subject of many investigations. The most adequate gene modification technique also remains to be established. Different transgenes have already been tested in animal models and transgenic mice. Several cell types were evaluated during the last decades and several vectors or transfection methods were analysed. From these essays, over time, several proofs of principles were made to demonstrate the feasibility of this type of therapy. For DMD, it is possible to express several truncated versions of dystrophin or exon skipping molecules. It is also possible to express other molecules that would mitigate the phenotype. Different cell types are also available. From the well documented myoblasts to the AC133 positive cells, the choice of cell types is exploding. Gene modification also evolved during the last decade. Efficient transfection technique and viral vectors are currently available. Given all these possibilities, the researcher has to make several choices. This review is trying to give clues of how to make those choices.

The C31 integrase system represents a novel technology that opens up new possibilities for gene therapy. The C31 integrase can integrate introduced plasmid DNA into preferred locations in unmodified mammalian genomes, resulting in robust, long-term expression of the integrated transgene. This review describes the nature of the integration reaction and the genomic integration sites used by the enzyme in human cells. Preclinical applications of the system to gene therapy to date are summarized, including in vivo use in liver, muscle, eye, and joint and ex vivo use in skin keratinocytes, muscle precursor cells, and T cell lines. The safety of this phage integrase system for gene therapy is evaluated, and its strengths and limitations are compared to other gene therapy approaches. Ongoing and planned improvements to the phage integrase system are discussed. We conclude that gene therapy strategies using C31 integrase and its derivatives offer great promise for success in the near term.

Conventional cancer treatments are often hampered by a lack of tumour selectivity, resulting in toxicity to healthy tissue. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy approach that aims to improve the selectivity of chemotherapy by enabling cancer cells to convert non-cytotoxic prodrugs to cytotoxic drugs. Many enzyme/ prodrug systems have been described, some of which have already been tested in clinical trials. A key component of GDEPT is a foreign enzyme that is expressed selectively at the tumour site where it converts the prodrug into the cytotoxic agent. The gene encoding the prodrug-activating enzyme needs to be expressed selectively and efficiently in tumour cells in order to spare normal tissue from damage. Substantial efforts have been made to develop gene therapy vectors that are capable of targeting cancer cells. A large number of gene delivery systems have been described for GDEPT: Viral vectors are the most advanced. They include replication-deficient and replication-selective (oncolytic) viruses. Recent advances in engineering viruses for GDEPT are reviewed in this article and data from both preclinical studies and clinical trials are discussed.

Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, even before any gene expression can occur. While many transfection strategies focus on DNA condensation and overcoming the plasma membrane, events associated with the intracellular trafficking of the DNA complexes have not been as extensively studied. Once internalized, plasmids must travel potentially long distances through the cytoplasm to reach their next barrier, the nuclear envelope. This review summarizes the current progress on the cytoplasmic trafficking and nuclear transport of plasmids used for gene therapy applications. Both of these processes utilize specific and defined mechanisms to facilitate movement of DNA complexes through the cell. The continued elucidation and exploitation of these mechanisms will lead to improved strategies for transfection and successful gene therapy.

Hematopoietic stem cells (HSCs) have unique properties of self-renewal, differentiation and proliferation. HSCs are easily accessible, and can be readily delivered back to patients by autologous transplantation, which renders them as attractive targets for ex vivo gene therapy. The adeno-associated virus (AAV) vectors have to date not been associated with any malignant disease, and have gained attention as a potentially safer alternative to the more commonly used retroviral vectors for HSC gene therapy. Although conflicting data exist with regard to HSC transduction by AAV vectors, in this review, we provide an overview of AAV-mediated HSC gene transfer - obstacles as well strategies to improve the transduction efficiency - and the potential use of AAV vectors for gene therapy of human diseases involving HSCs.