Download Chapter 9 Suicide Gene Therapy

‫‪Dr. S Hosseini-Asl‬‬
‫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.
 Unfortunately, an insufficient therapeutic index, a lack of
specificity, and the emergence of drug-resistant cell
subpopulations often hamper the efficacy of drug
therapies.
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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.
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 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
nonmalignant cells may be suitable targets.
and
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 The possibility of rendering cancer cells more sensitive
to drugs or toxins by introducing “suicide genes” has
two alternatives:
1.
Toxin gene therapy, in which the genes for toxic
products are transduced directly into tumor cells,
2. Enzyme-activating prodrug therapy, in which the
transgenes encode enzymes that activate specific
prodrugs to create toxic metabolites.
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 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.
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 The terms suicide gene therapy and GDEPT can be
used interchangeably to describe a two-step treatment
designed to treat solid tumors:
1. The gene for a foreign enzyme is delivered and
targeted in a variety of ways to the tumor where it is to
be expressed.
2. Prodrug is administered that is activated to the
corresponding drug by the foreign enzyme expressed
in the tumor.
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 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.
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
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variables include: ambiguity, cohesiveness and diffusion of responsibility.
The main advantages of optimized
suicide gene therapy systems:
1. Increased selectivity for cancer cells, reducing side effects.
2. Higher concentrations of active drug at the tumor,
compared to the concentrations accessible 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.
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The most important hurdles should
be overcome:
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.
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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.
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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), Damino-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.
1.
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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.
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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.
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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.
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 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.
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 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
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 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.
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Vectors in Suicide Gene Therapy
 Adenoviruses have achieved better transduction rates (10–50%)
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
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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.
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