Suicide Gene Therapy Methods and Reviews Edited by Caroline J. Springer M E T H O D S I N M O L E C U L A R M E D I C I N E TM Suicide Gene Therapy Methods and Reviews Edited by Caroline J. Springer Introduction to Suicide Gene Therapy 1 1 From: Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy: Methods and Reviews Edited by: C. J. Springer © Humana Press Inc., Totowa, NJ 1 Introduction to the Background, Principles, and State of the Art in Suicide Gene Therapy Ion Niculescu-Duvaz and Caroline J. Springer 1. 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. Unfortunately, an insufficient therapeutic index, a lack of specificity, and the emer- gence of drug-resistant cell subpopulations often hamper the efficacy of drug thera- pies. Despite the significant progress achieved by chemotherapy in the treatment of disseminated malignancies, the prognosis for solid tumors remains poor. A number of specific difficulties are associated with the treatment of solid tumors, where the 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 an- other major drawback. Attempts to target therapies to tumors have been addressed by using prodrugs activated in tumors by elevated selective enzymes and are described in Chapter 27. An alternative strategy that use antibodies to target tumors with foreign enzymes that subsequently activate prodrugs is described in Chapter 26. One approach aimed at enhancing the selectivity of cancer chemotherapy for solid tumors relies on the application of gene therapy technologies. Gene therapies are tech- niques for modifying the cellular genome for therapeutic benefit. In cancer gene therapy, both malignant and nonmalignant cells may be suitable targets. The possibil- ity of rendering cancer cells more sensitive to drugs or toxins by introducing “suicide genes” has two alternatives: toxin gene therapy, in which the genes for toxic products are transduced directly into tumor cells, and enzyme-activating prodrug therapy, in which the transgenes encode enzymes that activate specific prodrugs to create toxic metabolites. The latter approach, known as suicide gene therapy, gene-directed enzyme prodrug therapy (GDEPT) (1,2), virus-directed enzyme prodrug therapy 2 Niculescu-Duvaz and Springer (VDEPT) (3), or gene prodrug activation therapy (GPAT) (4) may be used, in isola- tion or combined with other strategies, to make a significant impact on cancer treat- ment. In this chapter, the terms GDEPT and suicide gene therapy are used. The terms suicide gene therapy and GDEPT can be used interchangeably to de- scribe a two-step treatment designed to treat solid tumors. In the first step, the gene for a foreign enzyme is delivered and targeted in a variety of ways to the tumor where it is to be expressed. In the second step, a prodrug is administered that is activated to the corresponding drug by the foreign enzyme expressed in the tumor. 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 (5). The main advantages of optimised suicide gene therapy systems are as follows: 1. Increased selectivity for cancer cells, reducing side effects. 2. Higher concentrations of active drug at the tumor, compared to the concentrations acces- sible by classical chemotherapy. 3. Bystander effects generated. 4. Tumor cell enzyme transduction and kill may induce immune responses that enhance the overall therapeutic response. 5. 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. A number of hurdles are still to be overcome. The most important are the following: 1. The vectors for gene transduction that target the tumor and achieve efficient infection of cancer cells. 2. Ideally, the vectors should be also nonimmunogenic and nontoxic. 3. The control of gene expression at the tumor. These issues will be addressed in this chapter and in Chapters 2–8 on vectors and should be read in conjunction with reviews on the background and principles of GDEPT (6–8), viral vectors (9–15), and nonviral vectors (16,17), the kinetics of activation (18), enzymes for GDEPT (19), the BE (20), and prodrugs designed for GDEPT (21–23). Herein we summarize the state of the art of suicide gene therapy highlighting recent progress and the areas that to date have hampered the development of suicide gene therapy. 2. 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 Introduction to Suicide Gene Therapy 3 from any circulating endogenous enzymes, and should be expressed in sufficient con- centration for therapeutic efficacy. The enzymes proposed for suicide gene therapy can be characterized into two major classes. 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. 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-glucu- ronidase (β-Glu), and cytochrome P450 (CYP). The advantages of such systems resides in the reduction of the potential for inducing an immune response. However, their pres- ence in normal tissues is likely to preclude specific activation of the prodrugs only in tumors unless the transfected enzymes are modified for different substrate requirements. The genes can be engineered to express their product either intracellularly or extra- cellularly in the recipient cells (1). The extracellularly expressed variants are either tethered to the outer cell membrane (1,24) (see also Chapter 14) or secreted from cells (see Chapter 15). There are potential advantages to each approach. Where the enzyme is intracellularly expressed, the prodrug must enter the cells for activation and, subse- quently, the active drug must diffuse through the interstitium across the cell mem- brane to elicit a BE. Cells in which the enzyme is expressed tethered to the outer surface or secreted are able to activate the prodrug extracellularly. A more substantial BE should therefore be generated in the latter system, but spread of the active drug into the general circulation is a possible disadvantage (1,24). The design of prodrugs tailored for GDEPT is described in depth in Chapter 9. The basic prodrug and drug requirements of a suicide gene therapy system are briefly described herein. Good pharmacological properties, good pharmacokinetic properties of prodrugs, low cytoxicity of prodrugs with high cytotoxicity of the activated drugs, and effective activation of prodrugs by the expressed enzyme are all important features. Prodrugs should be chemically stable under physiological conditions and be highly diffusible in the tumor interstitium. The released drugs should be as potent as possible, highly dif- fusible, ideally active in both proliferative and quiescent cells, and induce BEs. The activation of the prodrugs is a key step in suicide gene therapy. It is an advan- tage if the expressed enzyme can activate the prodrug directly to the drug, without the need for additional steps requiring further catalysis, because it is possible for the host endogenous enzymes needed for the latter steps to become defective or deficient in cancer cells. 4 Niculescu-Duvaz and Springer Two basic types of prodrug have been used in GDEPT: the directly linked and the self-immolative prodrugs. The directly linked prodrugs can be defined as a pharmaco- logical inactive derivative of a drug, which requires chemical transformation to release the active drug. In terms of anticancer activity, the conversion of the prodrug to an active drug results in a sharp increase in its cytotoxicity. In a directly linked prodrug, the active drug is released directly following the activation process (see Chapter 9). A self-immolative prodrug can be defined as a compound generating an unstable intermediate which, following the activation process, will extrude the active drug in a number of subsequent steps. The most important feature is that the site of activation is normally separated from the site of extrusion. The activation process remains an enzy- matic one. However, the extrusion of the active drug relies on a supplementary spon- taneous fragmentation. Potential advantages of self-immolative prodrugs are the possibility of altering the lipophilicity of the prodrugs with minimal effect on the acti- vation kinetics and the possibility to improve unfavorable kinetics of activation as a result of unsuitable electronic or steric features of the active drug. The range of drugs that can be converted to prodrugs is greatly extended and is unrestricted only by the structural substrate requirements for a given enzyme. A large number of enzyme–prodrug systems have been developed for GDEPT in the recent years and are summarized in Table 1. 2.1. Quantitative Data In order to compare different GDEPT systems in terms of therapeutic efficiency, each system should be characterized by relevant quantitative parameters. Some parameters refer to the activation process that can be described by kinetic parameters (K M , V max , and k cat ) (see Table 2). The concentration of the drug and the rate at which it is released at the activation site depends on the kinetic parameters of the enzyme– prodrug system. Often, published V max and K M values are not compared under equiva- lent conditions, whilst measuring the maximum velocity of the activation reaction and the concentration of substrate needed to reach half of this maximum velocity. Thus, there are insufficient data on enzyme–prodrug systems to allow GDEPT sys- tems to be compared. As a rule, however, a low K M and high V max (or k cat ) would be expected to favor the systems. The comparison of the yeast CD with bacterial CD bears out this prediction. The yeast enzyme, which proved to be more effective than its bacterial counterpart in GDEPT experiments, exhibits lower K M and higher V max than the bacterial homolog (see Table 2). Unfortunately, comparable values for the V max of these enzymes cannot be obtained because the V max has been determined un- der very different experimental conditions for the various systems and is expressed in different ways, making direct comparisons impossible. Despite these caveats, it ap- pears from the data in Table 2 that prodrugs such as CMDA (a substrate of CPG2), GCV (a substrate of HSV-TK), and CPT-11 (a substrate of CA) are superior to 5-FC (a substrate of CD) or 5'-FDUR (a substrate of TP) because the latter have high K M and low V max . The turnover number, k cat , provides additional information about the reaction rate, but the implications of this measure for tumor cell killing is unclear, because it is not yet known if sudden release of the active drug is more effective than Introduction to Suicide Gene Therapy 5 a slow, constant release or if quiescent and proliferating cells differ in their sensitiv- ity to drugs released at different rates. Two biological parameters can be use to compare the different GDEPT systems. These are the potential of activation of a given system and its degree of activation. The first parameter is defined as the ratio of the IC 50 of the prodrug to the IC 50 of the re- leased drug in a control nontransfected cell system. It represents the maximum possible efficiency of a given enzyme–prodrug system towards a cell line. The degree of activa- tion is defined as the ratio of the IC 50 of the prodrug in the nontransfected cell line to the IC 50 of the prodrug in the transfected or infected cell line and demonstrates the efficiency of the system in a cell line (18). These parameters allow a fair comparison between suicide gene therapy systems in vitro and should also be helpful in designing new systems. 2.2. New Systems Most of the GDEPT systems summarized in Table 2 are described comprehen- sively in this volume (see Chapters 9–15). However, a number of new systems have been reported in the last three years and will be briefly reviewed here. The horseradish peroxidase (HRP) enzyme/indole-3-acetic acid (IAA) prodrug sys- tem is described with the potential for hypoxia-regulated gene therapy (41). At physi- ological pH, IAA is activated by HRP to a long-lived species (radical) that is able to cross cell membranes, and has significantly increased cytotoxicity than the prodrug. This system is claimed to be active against T24 bladder carcinoma cells in vitro (41). Another recently developed system is CYP1A2/acetaminophen (37). Acetaminophen is converted to the chemically reactive metabolite N-acetyl-benzoquinoneimine (NABQI). Incubation of H1A2MZ cells with acetaminophen (4–20 mM) causes ex- tensive cytotoxicity. When 5% of cells expressing CYP1A2 were treated with acetami- nophen, complete cell killing resulted in 24 h. A potent BE was reported. Similar activity was described against the HCT116 colon carcinoma cells and SKOV-3 ova- rian cancer cells but not with MDA MB 361 cells, where a 50% transfection is required to achieve total cell kill (37). Tyrosinase has been investigated as a potential prodrug-activating enzyme for GDEPT. However, its use was hampered by the low expression of tyrosinase transgenes in nonmelanotic cells and by the low activity of the enzyme. Recently, mutants of tyrosinase, which accumulate in various cellular compartments (the wild- type enzyme is present only in melanosomes), overcome these difficulties. A GDEPT system, mutated tyrosinase/N-acetyl-4S-cysteaminyl phenol (NAcSCAP) or 4- hydroxyphenyl propanol (HPP), was recently developed. Expression of the mutated enzyme was induced by transfection of human tumor cells (9L gliosarcoma, MCF-7 breast adenocarcinoma, and HT-1080 fibrosarcoma). Further administration of NAcSCAP or HPP stopped cell proliferation and induced cell death in a dose- dependent manner (42). Escherichia coli uracil phosphoribosyl transferase (UPRT) (E.C. 2.4.2.9) (the homologs in human cells are orotate phosphoribosyl transferase [E.C. 2.4.2.10] or uridine-5'-monophosphate synthase) catalyzes the conversion of uracil to uridine-5'- monophosphate. This enzyme is also able to mediate the conversion of 5-FU into 5- 6 Niculescu-Duvaz and Springer 6 Table 1 Enzyme–Prodrug Systems System K M V max k cat no. Names and codes Origin Prodrugs Released (pro)drugs (µM)(nM/mg/min) (min –1 ) 1 Carboxyl esterase Human, Irinotecan SN-38 23–52.9 1.43 × 10 –3 — (CE) rabbit 7-ethyl-10-[4-(1- 7-ethyl-10-hydroxy- piperidino)-1- (20S)-camptothecin piperidino]- carbonyloxy-(20S)- camptothecin 2 Carboxypeptidase A Human MTX-α-peptides MTX 8.2.–96 — 12,250±1135 (CPA) 3 Carboxypeptidase G2 Pseudomonas CMDA CMBA, 3.4 — 34,980 (CPG2)(E.C.3.4.22.12) R16 ZD-2767P Phenol-bis-iodo 2.0 — 1,770 nitrogen mustard. Self-immolative Alkylating agents, prodrugs anthracycline antibiotics Introduction to Suicide Gene Therapy 7 7 4 Cytochrome P450 Human, Oxazaphosphorines: human CYP2B1, rat, cyclophosphamide Alkylating agents 300 39.1 — 2B6,2C8, 2C9, 2C18 rabbit (CP) ifosfamide (IF) and 3A Rat: CYP2B1 Ipomeanol, 2-aminoan- Toxic metabolites 480 17.8 — thracene (2-AA); Rabbit: CYP 4B1 Acetaminophen N-acetyl (with or without benzoquinone P450 reductases) imine (NABQI) 5 Cytosine deaminase E. coli, 5-Fluorocytosine 5-Fuorouracil 17,900 11.7 — (CD) E.C. 3.5.4.1 yeast (5-FC) (5-FU) 800 68 (with or without uracilphosphoribosyl transferase, UPRT) 6 D -Amino-acid oxidase Rohdoto- D -Alanine Hydrogen peroxide —— — (DAAO) rula gracilis, (yeast) 7 Deoxycytidine kinase, Human Cytosine arabinoside Cytosine arabinoside 25.6 — — (dCK), E.C.2.7.1.21 monophosphate 8 Niculescu-Duvaz and Springer 8 Table 1(continued) Enzyme–Prodrug Systems System K M V max k cat no. Names and codes Origin Prodrugs Released (pro)drugs (µM)(nM/mg/min) (min –1 ) 8 Deoxyribonucleotide Drosophila Analogs of Analogs of — — — kinase melanogaster pyrimidine and pyrimidine and (DmNK) purine purine 2-deoxynucleosides 2'-deoxynucleotide monophosphates 9 DT-Diaphorase Human, Bioreductive agents: Reduced forms — — — (DT-D) rat E09, etc. 10 β−Galatosidase E. coli Self-immolative Anthracycline — — — (β−Gal) E.C. 3.2.1.23 prodrugs from antibiotics anthracycline antibiotics 11 β-Glucuronidase Human Self-immolative Doxorubicin 10.2 39.4 — (β-Glu) HM-1826 12 Horseradish Plant Indole-3-acetic acid ?——— peroxidase (HPP) (IAA) 13 β−Lactamase Bacterial Self-immolative Alkylating agents, 160 — 3,300– (β-L) cephem prodrugs Vinca alkaloids, 72,000 anthracycline antibiotics 14 Methionine-α,γ–liase Pseudomonas Selenomethionine Methylselenol — — — (MET) putida Introduction to Suicide Gene Therapy 9 9 Table 1(continued) Enzyme–Prodrug Systems System K M V max k cat no. Names and codes Origin Prodrugs Released (pro)drugs (µM)(nM/mg/min) (min –1 ) 15 Multiple drug Tomato Acetylated 6-TG, 6-TG, MTX, — — — activating enzyme MTX, and other cytotoxic purines (MDAE) purines 16 Nitroreductase E. coli CB-1954 and Alkylating agents; 900 — 180 (NR) analogs; Self-immolative Alkylating agents prodrugs pyrazolidines, enediynes 17 Penicillin G amidase E. coli ———— — (PGA) 18 Purine nucleotide E. coli Purine nucleosides 6-methylpurine, 14–23 422–638 a — phosphorylase, 2-fluoroadenine (PNP), E.C. 2.4.2.1 19 Thymidine kinase Herpes Modified pyrimidine Monophosphate (TK) simplex nucleosides: nucleotide analogs E.C. 2.7.1.21 virus GCV, 11–15.8(47) 1.3–22 × 10 –3 ACV, 305–375 3–4 ×10 −4 valacyclovir, etc. 20 Thymidine kinase, Varicella- FIAU, 56 680 b — (TK) zoster purine nucleosides virus araM [...]... N., Velu, T., and Calberg-Bacq, C.-M (2000) The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy Cancer Gene Ther 7, 1456–1468 83 Kaneko, Y and Tsukamoto, A (1995) Gene therapy of hepatoma: bystander effect s and non-apoptotic cell death induced by thymidine kinase and ganciclovir... Robbins, P D and Ghivizzani, S C (1998) Viral vectors for gene therapy Pharm Ther 80, 35–47 12 Zhang, W W (1999) Development and application of adenoviral vectors for gene therapy of cancer Cancer Gene Ther 7, 113–138 Introduction to Suicide Gene Therapy 23 13 Curiel, D T., Gerritsen, W R and Krul, M R (2000) Progress in cancer gene therapy Cancer Gene Ther 7, 1197–1199 14 Roth, M G and Curiel, D... Introduction to Suicide Gene Therapy 21 92% of animals This study clearly demonstrated that the distant BE was the result of an immune response (85) 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... Springer Vectors in Suicide Gene Therapy 29 2 Introduction to Vectors for Suicide Gene Therapy Caroline J Springer 1 Introduction Suicide gene therapy requires vectors or vehicles capable of efficient and selective gene delivery of the therapeutic genes to tumor cells A number of vector systems has been proposed for gene therapy These include: the viral vectors, adenoviruses (see Chapters 4 and 5), adeno-associated... in cancer gene therapy: principles and progress Anti-Cancer Drug Des 12, 275–304 18 Springer, C J and Niculescu-Duvaz, I (2000) Prodrug-activating systems in suicide gene therapy J Clin Investig 105, 1161–1167 19 Encell, L P., Landis, D M and Loeb, L A (1999) Improving enzymes for gene therapy Nature Biotechnol 17, 143–147 20 Mesnil, M and Yamasachi, H (2000) Bystander effect in herpes simplex virus–thymidine... granulocyte–macrophage colony-stimulating factor gene therapy induces complete tumor regression and generates antitumor immunity Cancer Gene Ther 7, 1519–1528 27 Walling, H W., Swarthout, G T., and Culver, K W (2000) Bystander-mediated regression of osteosarcoma via retroviral transfer of the herpes simplex virus thymidine kinase and human interleukin-2 genes Cancer Gene Ther 7, 187–196 28 Howard, B D., Boenicke,... carboxypeptidase G2 suicide gene therapy and the prodrug CMDA are due to a bystander effect Human Gene Ther 11, 285–292 Greco, O., Folkes, L K., Wardman, P., Tozer, G M., and Dachs, G U (2000) Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid Cancer Gene Ther 7, 1414–1420 Simonova, M., Wall, A., Weissleder, R., and Bogdanov, A (2000)... ifosfamide and 5-fluorocytosine Cancer Gene Ther 7, 629–636 52 Rogulski, K R., Wing, M S., Paielli, D L., Gilbert, J D., Kim J H., and Freytag, S O (2000) Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization Human Gene Ther 11, 67–76 53 Toda, M., Martuza, R L., and Rabkin, S D (2001) Combination suicide/ cytokine... transfection of suicide gene( s) together with genes able to increase the sensitivity of the tumors to radiation or enhance the potential of the host immune system with cytokine genes Improvements are needed in vector design area to enhance targeting and delivery of suicide genes Multiple options are available, including nonviral vectors, more complex systems involving coexpression of suicide genes with... UPRT, uracil phosphoribosyltransferase; see also Table 1 Niculescu-Duvaz and Springer NR/CB1954 14 12 14 Table 2 (continued) Bystander Effect Introduction to Suicide Gene Therapy 15 2.3.2 Multiple -Gene Transfection A different strategy to develop more efficient suicide gene therapy systems uses transgenes with greater than one gene Several different approaches have been reported Some prodrugs are activated . Suicide Gene Therapy Methods and Reviews Edited by Caroline J. Springer M E T H O D S I N M O L E C U L A R M E D I C I N E TM Suicide Gene Therapy Methods and Reviews Edited by Caroline. Reviews Edited by Caroline J. Springer Introduction to Suicide Gene Therapy 1 1 From: Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy: Methods and Reviews Edited by: C. J. Springer © Humana. Background, Principles, and State of the Art in Suicide Gene Therapy Ion Niculescu-Duvaz and Caroline J. Springer 1. Introduction to the Background and Principles of Suicide Gene Therapy Chemotherapy