Two basic strategies exist for cancer gene therapy. The first conceived strategy is ''ex vivo'' gene therapy in which a tumor or fibrous tissue biopsy is taken from a cancer patient whereupon individual tumor cells or fibroblasts are isolated and grown in vitro (Fig. 3). Therapeutic genes are then inserted into these cells typically using retroviral vector infection in tissue culture. The cells are subsequently irradiated and then reimplanted into the original tumor site or distant to the tumor site by autologous transplantation. The level of irradiation is controlled so as not to immediately kill the cells but to prevent growth and allow only a short period of survival after reimplantation. This strategy has been more commonly applied to classic cancer vaccine and cytokine gene therapy strategies. Although this approach is feasible with suicide gene therapy, the predominant tumor response would come from a metabolic cooperation or immune bystander effect subsequent to prodrug
administration and killing of the reimplanted transduced tumor cells. The inefficiency of such a system in general supports the predominant research and clinical focus on in vivo approaches for suicide gene therapy.
The second is basic strategy for cancer gene therapy is ''in vivo'' gene therapy where DNA, viral vectors alone, or packaging cell lines producing viral vectors are administered directly to a cancer patient (Fig. 4). The most common route of delivery is via direct injection of the delivery vehicle and gene or the packaging cell line into the tumor. Systemic injection is possible, but with present technology, results in limited exposure of the tumors to the vehicle. Tumors within tissues that have a large amount of blood flow or act as filters such as the lung or liver may prove more amenable to systemic delivery. As gene transfer technology continues to advance, the development of vehicles with tumor- or tissue-specific receptor uptake or promotor activity may allow for systemic administration of the DNA or viral vector carrying the suicide gene.
A. REPLICATION-COMPETENT VIRAL THERAPY: A FORM OF ''SUICIDE THERAPY''
Until very recently, viral vectors used in all gene therapy strategies were replication incompetent. Although this design was intended to introduce safety measures, it does not take advantage of the powerful ability of viruses to infect target cells and replicate and release viral particles, thereby killing target cells and spreading to surrounding target cells. Continued research and investigation in the fields of molecular biology and the genetics of cancer have led to a greater under
standing of the principles of viral replication and the genetics of carcinogenesis. This greater understanding has allowed the development of replication-selective oncolytic viruses for use as novel anticancer therapies.
The first replication-selective viral vector to move from preclinical studies to human cancer clinical trials was the Onyx-O15 adenovirus, also known as dl1520 (81,82). The key alteration that made this adenovirus replication selective was the deletion of the gene that codes for the p53-binding protein, E1B-55kDa. Typically, the adenovirus achieves replication in part through a process by which the E1B-55kDa protein binds a host cells p53, thereby allowing the cell to enter the S-phase of cell-cycle activity. The dl1520 adenovirus with its deletion of the E1B gene will not express the E1B-55kDa p53-binding protein upon infection of a target cell. The lack of E1B-55kDa expression will inhibit viral replication. However, in target cells that lack normal p53 expression, the dl1520 virus will maintain its ability to replicate, lyse a target cell, and spread to nearby cells. Because the majority of cancers have a loss of normal p53 function, cancer cells are the ideal target for a E1B-deleted replication-selective adenovirus therapy. There are many other evolving strategies for oncolytic viral vector therapy, and it is not the purpose of this chapter to discuss the present state of this novel therapy in detail. However, it is important to mention replication-selective oncolytic viral therapy because it may be thought of as a type of gene-dependent suicide therapy.
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