Dr. S Hosseini-Asl 1 به جای انکه به تاریکی لعنت بفرستید, یک شمع روشن کنید. کنفسیوس

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Introduction to the Background and Principles of Suicide Gene Therapy Chemotherapy is widely used with surgery and radiotherapy for the treatment of malignant disease. Selectivity of most drugs for malignant cells remains elusive. insufficienttherapeuticindexlackof specificityemergenceof drug-resistant cell subpopulations Unfortunately, an insufficient therapeutic index, a lack of specificity, and the emergence of drug-resistant cell subpopulations often hamper the efficacy of drug therapies. 3

A number of specific difficulties are associated with the treatment of solid tumors: Access of drugs to cancer cells is often limited by poor, unequal vascularization and areas of necrosis. The histological heterogeneity of the cell population within the tumor is another major drawback. Attempts to target therapies to tumors have been addressed by using prodrugs activated in tumors by elevated selective enzymes. An alternative strategy is using antibodies to target tumors with foreign enzymes that subsequently activate prodrugs. 4

enhancing the selectivity One approach aimed at enhancing the selectivity of cancer chemotherapy for solid tumors relies on the application of gene therapy technologies. Gene therapies are techniques for modifying the cellular genome for therapeutic benefit. In cancer gene therapy, both malignant and nonmalignant cells may be suitable targets. 5

The possibility of rendering cancer cells more sensitive to drugs or toxins by introducing “suicide genes” has two alternatives: 1. Toxin gene therapy 1. Toxin gene therapy, in which the genes for toxic products are transduced directly into tumor cells, 2. Enzyme-activating prodrug therapy 2. Enzyme-activating prodrug therapy, in which the transgenes encode enzymes that activate specific prodrugs to create toxic metabolites. 6

The latter approach, known as suicide gene therapy, gene-directed enzyme prodrug therapy (GDEPT), virus-directed enzyme prodrug therapy (VDEPT), or gene prodrug activation therapy (GPAT) may be used, in isolation or combined with other strategies, to make a significant impact on cancer treatment. 7

The terms suicide gene therapy and GDEPT can be used interchangeably to describe a two-step treatment designed to treat solid tumors: The gene for a foreign enzyme is delivered and targeted in a variety of ways to the tumor where it is to be expressed Prodrug is administered that is activated to the corresponding drug by the foreign enzyme expressed in the tumor. 8

Ideally Ideally, the gene for the enzyme should be expressed exclusively in the tumor cells compared to normal tissues and blood. The enzyme must reach a concentration sufficient to activate the prodrug for clinical benefit. The catalytic activity of the expressed protein must be adequate to activate the prodrug under physiological conditions. Because expression of the foreign enzymes will not occur in all cells of a targeted tumor in vivo, a bystander effect (BE) is required, whereby the prodrug is cleaved to an active drug that kills not only the tumor cells in which it is formed but also neighboring tumor cells that do not express the foreign enzyme. 9 The bystander effect is a social psychological phenomenon that refers to cases in which individuals do not offer help to a victim. The probability of help is inversely related to the number of bystanders. In other words, the greater the number of bystanders, the less likely it is that any one of them will help. Several variables help to explain why the bystander effect occurs. These variables include: ambiguity, cohesiveness and diffusion of responsibility.

The main advantages of optimized suicide gene therapy systems: Increased selectivity for cancer cells, reducing side effects Higher concentrations of active drug at the tumor, compared to the concentrations accessible by classical chemotherapy Bystander effects generated Tumor cell enzyme transduction and kill may induce immune responses that enhance the overall therapeutic response Prodrugs are not required to exhibit intrinsic specificity for cancer cells; they are designed to be activated by the foreign enzymes, which is technically easier to achieve. 10

The most important hurdles should be overcome: The vectors for gene transduction that target the tumor and achieve efficient infection of cancer cells Ideally, the vectors should be also nonimmunogenic and nontoxic The control of gene expression at the tumor. 11

Enzymes and Prodrugs Used in Suicide Gene Therapy Systems There are specific requirements of the enzymes used in GDEPT. They should have high catalytic activity (preferably without the need for cofactors), Should be different from any circulating endogenous enzymes, and Should be expressed in sufficient concentration for therapeutic efficacy. 12

The enzymes proposed for suicide gene therapy can be characterized into two major classes: 13

1. The first class comprise enzymes of nonmammalian origin with or without human counterparts. Examples include viral thymidine kinase (TK), bacterial cytosine deaminase (CD), bacterial carboxypeptidase G2 (CPG2), purine nucleotide phosphorylase (PNP), thymidine phosphorylase (TP), nitroreductase (NR), D- amino-acid oxidase (DAAO), xanthine–guanine phosphoribosyl transferase (XGPRT), penicillin-G amidase (PGA), β-lactamase (β-L), multiple-drug activation enzyme (MDAE), β-galactosidase (β-Gal), horseradish peroxidase (HRP), and deoxyribonucleotide kinase (DRNK). Those enzymes that do have human homologs have different structural requirements with respect to their substrates in comparison to the human counterparts. Their main drawback is that they are likely to be immunogenic. 14

The second class of enzymes for suicide gene therapy comprises enzymes of human origin that are absent from or are expressed only at low concentrations in tumor cells. 2. The second class of enzymes for suicide gene therapy comprises enzymes of human origin that are absent from or are expressed only at low concentrations in tumor cells. Examples include deoxycytidine kinase (dCK), carboxypeptidase A (CPA), β-glucuronidase (β-Glu), and cytochrome P450 (CYP). The advantages of such systems resides in the reduction of the potential for inducing an immune response. 15

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Improved Strategies 1. Mutation of the Enzymes 2. Multiple-Gene Transfection 3. New Prodrugs for Old Systems A different way of improving GDEPT systems is to design prodrugs with lower cytotoxicity. Also, complementary strategies of increasing the cytotoxicity of the released drugs or improving the activation process may be helpful 4. Potentiation and Synergistic Effects An alternative strategy to increase the efficiency of GDEPT systems was developed from a better understanding of the mechanisms of action of the released drugs based on synergistic or additive effects of compounds. This approach was applied to improve the HSV-TK/GCV and CD/5-FC systems 5. Radiosensitization Radiotherapy is a valuable alternative to chemotherapy with or without surgery, in the complex strategy of cancer treatment. Therefore, its combination with suicide gene therapy has been proposed as an advantage. HSV-TK gene transfection was used to increase the radiosensitivity of various cell lines. 20

Immune Effect in Suicide Gene Therapy Systems It is generally accepted that the immune response improves the efficacy of GDEPT systems in vivo. Several lines of evidence strengthen this view. 21

first The first is that although the BE has been observed in immunocompromised animals, data suggest that the BE in vivo is mediated largely through the release of cytokines and, therefore, GDEPT systems are more efficient in immunocompetent animals. 22

second The second is the existence of the “distant bystander effect.” A distant BE has been reported in a number of situations when tumors anatomically separated and with no possible metabolic cooperation were inhibited after suicide gene therapy was administered only to one tumor 23

third The third line of evidence is given by the cotransfection of both suicide genes and immune enhancing genes. The transgenes containing both a suicide gene and granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin (IL) gene proved to be more effective when compared to the suicide gene therapy alone. 24

Vectors in Suicide Gene Therapy Adenoviruses have achieved better transduction rates (10–50%) in vivo than retroviruses (0.9–14.6%). Nonviral vectors (with electroporation) have achieved up to 8% transfection in vivo. Unusually high values (up to 59%) have been reported for nonviral vector transfection in vivo. However, the highest values (>80%) were reported for a combination of viral and nonviral vectors (adenovirus complexed with PEI or DEAE-dextran) For applications such as ex vivo infections, direct administration of the vector to target tissues in vivo, or locoregional delivery, the ability to target specific cells may not be necessary. Of 333 protocols that were ongoing in cancer gene therapy in February 2001, >38% used intratumoral or locoregional delivery. However, if systemic delivery is required, targeting will be of major importance. 25

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