Suicide Gene Therapy Strategies

A key issue in the success of a suicide gene therapy strategy is the interaction between the enzyme produced and the prodrug administered. The selection of the enzyme and prodrug combination is influenced by certain variables critical to enyzme-substrate kinetics. Two important variables exist for the enzyme. The first is the speed of activation of the prodrug. The most effective suicide genes will express enzymes that rapidly activate the prodrug. Enzymes that are slower in their activation will be dependent on either higher concentrations of the prodrug or prolonged administration. Issues such as half-life and intracellular degradation and clearance mechanisms will also limit the presence of the prodrug. The second variable is the efficiency of prodrug activation. Enzymes that are highly efficient in converting the prodrug substrate into its toxic metabolite should prove advantageous because of known variances in the levels of effective gene transfer and gene expression (enzyme production) inherent to in vivo suicide gene transfer. Regarding the prodrug, it should be at least 100 times more cytotoxic than the preactivated form upon enzyme activation (7). Because many different types of prodrugs can be designed to achieve maximal activation, the most important and probably most limiting factor is the enzyme and therefore choice of suicide gene. Table 1 depicts the more commonly used suicide genes and corresponding prodrug combinations under investigation for gene therapy application. Table 2 shows the mechanisms of toxicity for selected genes that are presently being investigated in both preclinical and clinical studies.

Despite choosing a generally efficient enzyme-prodrug combination that proves successful with in vitro experimentation, there may not be a paralleled success after the transition to in vivo application against established tumors. In vivo effi-

Table l Selected Suicide Gene Therapy Strategies

Gene/enzyme

Prodrug

Initial toxic metabolite

Herpes simples virus thymidine kinase (HSV-tk) Cytosine deaminase (CD) Varicella zoster virus thymidine kinase (VZV-tk) Escherichia coli nitroreductase (NTR) Cytochrome P450 B1

(CYP2B1) Carboxypeptidase G2 (CPG2)

Ganciclovir (GCV)

5-Fluorocytosine (5-FC)

6-Methoxypurine arabinonucleoside (araM)

5-(Aziridin-1-yl)-2, 4-dinitrobenzamide (CB 1954)

Cyclophosphamide (CPA)

Benzoic acid mustard gluonuridebenzoic acid mustard (CMDA)

6-Thioxanthine (6-TX)

6-Methyl purine deopxyriobside (MePdR)

Ganciclovir monophosphate

5-Fluorouracil (5-FU)

Adenine arabinonucleoside monophosphate

5-(Aziridin-1-yl)-4-hydroxyamino-2-

nitrobenzamide 4-Hydroxy-cyclophosphamide (4-HCPA)

6-Thioxanthine monophosphate (6-XMP) 6-Methylpurine (MeP)

cacy may also vary among different types or classes of tumors for any one given enzyme-prodrug combination. An example of this is found within the HSV-tk gene therapy strategy, the most widely used suicide gene in both preclinical investigation and human clinical trials. Although HSV-tk has proven effective in many different solid tumors, it is generally less effective against hematopoietic malignancies (8). It has been hypothesized that HSV-tk is down-regulated or lost more quickly in hematopoietic tumors such as leukemia, which results in ineffective or insufficient conversion of the prodrug to achieve antitumor effects (9).

HSV-tk has shown the greatest potential to date for human application across a broad range of malignancies, and for this reason it is the most popular and widely studied suicide gene therapy strategy. The importance of HSV-tk gene transfer centers on its ability to render cells sensitive to the acyclic guano-

sine analog ganciclovir (GCV) (10,11). HSV-tk is a prototype ''suicide gene'' because it encodes a viral enzyme that is foreign to mammalian cells and will convert an inactive and relatively nontoxic prodrug to a toxic product. Upon effective HSV-tk gene transfer and expression, the prodrug GCV is monophosphorylated by the enzyme. Intracellular host kinases then metabolize this monophosphorylated nucleoside analog into di- and triphosphates (12). The triphosphate form of GCV is then incorporated into the replicating DNA chain in dividing cells and inhibits DNA polymerase. Inhibition of DNA poly-merase results in chain termination, disruption of DNA synthesis, and cell death. The phosporylation of GCV impairs its ability to cross the cell membrane and, as a result, the halflife increases by 6-fold to 18 to 24 h (13,14). The extended half-life of the phosphorylated GCV strengthens the overall anticancer effect of this HSV-tk strategy. With respect to sensitivity, viral thymidine kinase is approximately 1000 times more efficient in phosphorylating GCV than its mammalian counterpart (13). Because GCV is an excellent substrate for

Table 2

Mechanisms of Cytotoxicity of Select Suicide Gene Therapy Strategies

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