Current Gene Therapy - Volume 2, Issue 3, 2002

One of the most rapidly advancing areas of gene therapy is vector development. For the majority of gene therapy procedures, efficient and selective transduction would provide safe and more effective treatments at optimal vector doses. Advances in vector targeting strategies have been rapid within the field of DNA-based viruses, particularly adenovirus (Ad) and more recently adeno-associated virus (AAV) based vectors. Vector targeting at the level of virus: cell interaction can be achieved using both non-genetic and genetic methodology. Non-genetic approaches typically utilise bispecific antibodies that both neutralise wild-type virus tropism and provide a new cell binding capacity. For genetic targeting strategies, the virus capsid can be engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the virus'natural tropism. This review covers technological advances that have led to targeting of Ad and AAV and highlights the potential for these ‘designer’ viruses for future gene-based therapeutics.

Since Cystic Fibrosis (CF) is an autosomal recessive disorder due to mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, studies towards a gene therapy approach to its treatment followed immediately upon the cloning of the gene. It was demonstrated that the insertion of a single copy of the wild-type gene restored the normal phenotype in CF cells in vitro. Encouraging results were obtained in many in vivo model systems (CF transgenic mice) involving viral as well as non-viral vectors, which demonstrated the recovery of CFTR function in the airways. These results constituted the basis for human studies. Of those with a non-viral approach, a total of seven clinical trials using cationic lipids have reported data on efficiency, efficacy and safety. An effective gene transfer approach for the treatment of CF lung disease is not however imminent: low transfection efficiency and poor maintenance of gene expression are so far the main obstacles on this therapeutic path. On the other hand, no important adverse effects have been documented and repeated administration in humans is possible.The understanding of tissue and cellular barriers is a prerequisite for the development of more efficient non-viral gene therapy protocols for CF patients. While cationic lipids have been shown to be blocked by the mucous airway barrier and not be able to transfect differentiated respiratory epithelial cells, a new class of non-viral vectors, cationic polymers, are endowed with chemical and biological properties that make them more efficient in mediating gene transfer than lipids. Cationic polymers, such as polyethylenimine, are promising vectors for CF lung gene therapy.

Despite the development of new therapeutic strategies, cancer remains incurable in most patients with advanced disease. A recent potential improvement in therapeutic strategies is the concept of suicide gene therapy. After transfection with a suicide gene, cells can convert a harmless prodrug into its toxic metabolite, resulting in selective elimination of these cells. One of the most frequently studied therapeutic strategies is based on transfection with the herpes simplex virus thymidine kinase gene (HSV-tk), followed by ganciclovir administration. Despite promising results in vitro and in vivo, the antitumor effect in clinical trials remains poor, due to very low transfection efficiency. However, high percentages of transfected cells are not mandatory for complete eradication of a tumor in vivo. Transfected tumor cells appear to be capable of inducing the death of neighboring untransfected cells. This cell kill is called the “bystander effect”. Various attempts have been made to increase this effect. A substantial bystander effect could overcome the limitations of low transfection efficiency and result in an enhanced and possibly clinically worthwhile antitumor effect in patients. This review is focused on the HSV-tk / GCV system and gives an overview of current knowledge on the bystander effect in vitro and in vivo. In addition, theories concerning its mechanisms and possible approaches to augment this effect are discussed. Finally, we give an overview of clinical trials using suicide gene therapy.

Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery vectors, in particular for cancer gene therapy applications. In this review, we summarize various strategies used to selectively modify the natural tropism of recombinant adenoviruses. The advantages, limitations and potential impact on gene therapy operations of such modified vectors are discussed.

Autoimmunity accounts for a significant percentage of human disease and remains a challenging syndrome to treat. While systemic immunosuppression can be beneficial, the associated toxicity of the pharmacologic agents necessitates an antigen-specific approach to silence, eradicate or prevent the genesis of autoreactive immune cells. Gene therapy offers the possibility of providing precise antigen-targeted therapies, thereby sparing the patient the significant toxicity associated with lifelong commitment to chemical immunosuppressives. Gene-based therapies could include, but are not limited to the manipulation of immune networks of tolerance by antigen presentig cell engineering, proinflammatory cytokine blockade using soluble antagonists expressed from viral vectors as well as modulation of immune regulatory networks. The potential utility of gene therapy strategies promoting tolerance in two model autoimmune disorders, type I diabetes mellitus and rheumatoid arthritis are discussed in this review.

Traditionally, vectors for gene transfer / therapy experiments were mono- or bicistronic. In the latter case, vectors express the gene of interest coupled with a marker gene. An increasing demand for more complex polycistronic vectors has arisen in recent years to obtain complex gene transfer / therapy effects. In particular, this demand is stimulated by the hope of a more powerful effect from combined gene therapy than from single gene therapy in a process whose parallels lie in the multi-drug combined therapies for cancer or AIDS. In the 1980's we had only splicing signals and internal promoters to construct such vectors: now a new set of biotechnological tools enables us to design new and more reliable bicistronic and polycistronic vectors.This article focuses on the description and comparison of the strategies for co-expression of two genes in bicistronic vectors, from the oldest to the more recently described: internal promoters, splicing, reinitiation, IRES, self-processing peptides (e.g. foot-and-mouth disease virus 2A), proteolytic cleavable sites (e.g. fusagen) and fusion of genes. I propose a classification of these strategies based upon either the use of multiple transcripts (with transcriptional mechanisms), or single transcripts (using translational / post-translational mechanisms).I also examine the different attempts to utilise these strategies in the construction of polycistronic vectors and the main problems encountered. Several potential uses of these polycistronic vectors, both in basic research and in therapy-focused applications, are discussed.The importance of the study of viral gene expression strategies and the need to transfer this knowledge to vector design is highlighted.

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the β-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.