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Introduction to Suicide Gene Therapy 1
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
From: Methods in Molecular Medicine, Vol. 90, Suicide Gene Therapy: Methods and Reviews
Edited by: C. J. Springer © Humana Press Inc., Totowa, NJ
1
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 , V , and k ) (see Table 2). The concentration of the drug and the rate at which
M max cat
it is released at the activation site depends on the kinetic parameters of the enzyme–
prodrug system. Often, published V and K values are not compared under equiva-
max M
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 and high V (or k ) would be
M max cat
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 and higher V
M max
than the bacterial homolog (see Table 2). Unfortunately, comparable values for the
V of these enzymes cannot be obtained because the V has been determined un-
max max
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 . The turnover number, k , provides additional information about the
max cat
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 of the prodrug to the IC of the re-
50 50
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 of the prodrug in the nontransfected cell line to
50
the IC of the prodrug in the transfected or infected cell line and demonstrates the
50
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
Table 1
Enzyme–Prodrug Systems
System K V k
M max cat
no. Names and codes Origin Prodrugs Released (pro)drugs (µM) (nM/mg/min) (min–1)
6
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
N
piperidino]- ic
carbonyloxy-(20S)- u
camptothecin les
2 Carboxypeptidase A Human MTX-α-peptides MTX 8.2.–96 — 12,250±1135 cu
-
(CPA) D
u
v
a
3 Carboxypeptidase G2 Pseudomonas CMDA CMBA, 3.4 — 34,980 z
(CPG2)(E.C.3.4.22.12) R16 ZD-2767P Phenol-bis-iodo 2.0 — 1,770 an
nitrogen mustard. d S
Self-immolative Alkylating agents, p
r
prodrugs anthracycline in
g
antibiotics e
r
In
4 Cytochrome P450 Human, Oxazaphosphorines: tro
human CYP2B1, rat, cyclophosphamide Alkylating agents 300 39.1 — d
u
2B6,2C8, 2C9, 2C18 rabbit (CP) ifosfamide (IF) c
and 3A tio
n
Rat: CYP2B1 Ipomeanol, 2-aminoan- Toxic metabolites 480 17.8 — to
thracene (2-AA); S
u
ic
Rabbit: CYP 4B1 Acetaminophen N-acetyl id
e
(with or without benzoquinone G
P450 reductases) imine (NABQI) e
n
5 Cytosine deaminase E. coli, 5-Fluorocytosine 5-Fuorouracil 17,900 11.7 — e
(CD) E.C. 3.5.4.1 yeast (5-FC) (5-FU) 800 68 T
h
(with or without er
a
uracilphosphoribosyl p
y
transferase, UPRT)
7
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
7
8
Table 1(continued)
Enzyme–Prodrug Systems
System K V k
M max 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
8 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
N
anthracycline ic
antibiotics u
11 β-Glucuronidase Human Self-immolative Doxorubicin 10.2 39.4 — les
(β-Glu) HM-1826 cu
-
12 Horseradish Plant Indole-3-acetic acid ? — — — D
u
peroxidase (HPP) (IAA) v
13 β−Lactamase Bacterial Self-immolative Alkylating agents, 160 — 3,300– az
(β-L) cephem prodrugs Vinca alkaloids, 72,000 an
anthracycline d S
antibiotics p
r
14 Methionine-α,γ–liase Pseudomonas Selenomethionine Methylselenol — — — in
g
(MET) putida e
r
Table 1(continued) In
Enzyme–Prodrug Systems tro
d
u
System KM Vmax kcat c
no. Names and codes Origin Prodrugs Released (pro)drugs (µM) (nM/mg/min) (min–1) tio
n
to
15 Multiple drug Tomato Acetylated 6-TG, 6-TG, MTX, — — — S
activating enzyme MTX, and other cytotoxic purines u
ic
(MDAE) purines id
16 Nitroreductase E. coli CB-1954 and Alkylating agents; 900 — 180 e G
(NR) analogs; e
n
Self-immolative Alkylating agents e
prodrugs pyrazolidines, T
h
enediynes e
r
17 Penicillin G amidase E. coli — — — — — ap
(PGA) y
9 18 Purine nucleotide E. coli Purine nucleosides 6-methylpurine, 14–23 422–638a —
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 680b —
(TK) zoster purine nucleosides
virus araM
9
