Suicide gene therapy

October 2020
Presenter:
Parisa Ghasemiyeh
Pharm.D., PhD. Student of Pharmacotherapy,
School of Pharmacy,
Shiraz University of Medical Sciences,
Shiraz, Iran.
Suicide Gene Therapy for Cancer

Suicide gene therapy is based on the delivery of a gene encoding a
cytotoxic protein into tumor cells .
For this, there are two possible strategies:
1. Indirect gene therapy using enzyme-activated pro-drug, which
allows the conversion of a pro-drug into a lethal drug into cells.
2. Direct gene therapy using a toxin gene, whose expression can
change the stability of the cell membrane and reduce the viability of
tumor cells, or correct mutated pro-apoptotic genes, generally tumor
suppressor genes that in normal condition induce cell suicide.
Suicide gene therapy for cancer

Enzyme/Pro-drug Systems
Toxins
Pro-apoptotic Genes
Suicide gene therapy
1
2
3

Gene-directed enzyme pro-drug therapy (GDEPT):
A two-step treatment designed to treat solid tumors.
I. 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.
II. In the second step, a pro-drug is administered that is activated to
the corresponding drug by the foreign enzyme expressed in the
tumor.
1

Advantages of suicide gene therapy
1
• Increased selectivity for cancer cells.
2
• Reducing side effects.
3
• Higher concentrations of active drug at the tumor, compared to the concentrations accessible by
classical chemotherapy.
4
• Bystander effects generated.
5
• Tumor cell enzyme transduction and kill may induce immune responses that enhance the overall
therapeutic response.
6
• Pro-drugs 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.

Hurdles of suicide gene therapy
1
• The vectors for gene transduction that target the tumor and achieve
efficient infection of cancer cells.
2
• Ideally, the vectors should be also non-immunogenic and non-toxic.
3
• The control of gene expression at the tumor.

One of the most frequently studied therapeutic strategies is based on the
transfection of herpes simplex virus thymidine kinase (HSV-TK)
gene with ganciclovir (GCV) administration.
HSV-TK converts the antiviral drug GCV to a monophosphorylated
molecule that is then metabolized to the toxic triphosphate form by
cellular kinases, inhibiting DNA synthesis and producing cell death.
1

Another system consists in the combined administration of the cytosine
deaminase (CD) (enzyme found in bacteria and fungi, but absent in
mammalian cells) with 5-fluorocytosine (5-FC). This system relies on
the ability of CD to convert the nontoxic 5-FC into 5-fluorouracil (5-
FU), a potent cytotoxic chemotherapeutical, which is then transformed
by cellular enzymes, into potent pyrimidine antimetabolites (5-FdUMP,
5-FdUTP, 5-FUTP). CD/5-FC causes inhibition of cell proliferation and
cell death.
1

Another system employs the conversion of cyclophosphamide and
ifosfamide prodrug by human CYP450 isoforms into the unstable
metabolites 4-hydroxy forms that decay into phosphoramide mustard
and acrolein. These alkylating agents interfere with the DNA and the
strand breaks during DNA replication causing cell death.
1

An advantage of genetic prodrug activation therapy is the bystander
effect, because the prodrug has efficacy not only on transfected cells
but also on neighboring non-transfected cells.
1

 One alternative in suicide gene therapy for cancer is the use of a toxin
gene.
Advantages:
Toxins
2
• The presence of a prodrug is not required.1
• The toxicity of metabolites is reduced.2
• Has not limited the bioavailability due to the
lack of prodrug.3

Bacterial toxins
Immunotoxins
Toxins
2

1. Diphtheria toxin (Corynebacterium diphtheriae)  protein
synthesis inhibition.
Diphtheria toxin:
inactivates elongation factor 2 (EF-2)
by adenine diphosphate ribosylation (ADP-ribose)
and inhibits protein translation,
thereby triggering apoptosis.
A. Bacterial Toxins
2

2. Streptolysin O (SLO) is a membrane-damaging protein secreted by
bacteria from the genus Streptococcus.
It has been patented that the administration of a recombinant adenovirus
encoding SLO induced cell death in transfected cancer cells,
by forming pore and permeabilization of the cellular membrane.
A. Bacterial Toxins
2

3. Pseudomonas exotoxin induces cell killing by apoptosis through
adenosine diphosphate ribosylation and by inactivation of EF-2 and
thus inhibition of protein synthesis.
A. Bacterial Toxins
2

4. Several novel E. coli with toxin–antitoxin regulatory mechanisms
have been recently discovered.
A. Bacterial Toxins
2

1. MazF is a toxin that is counteracted by the MazE antitoxin.
-MazF exhibits sequence-specific ribonuclease activity toward single-
or double-stranded RNA regions,
-Results in degradation of cellular mRNA.
-Causes global translation inhibition.
-Thereby effectively inhibiting cellular protein synthesis.
-Thus inhibiting cell growth.
B. Immunotoxins
2

2. Apoptin, the VP3 protein from chicken anemia virus (CAV), which
induces death in tumor cells but not in normal cells.
Apoptin is phosphorylated and translocates to the nucleus, enabling its
cytotoxic activity.
B. Immunotoxins
2

• The corrective gene therapy of genes involved in cell death and
apoptosis, which induce cell suicide, also has been developed.
• Mutations of p53 gene are the most frequent abnormality identified in
human tumors and numerous studies have shown that restoring p53
function can induce apoptosis in cancer cells.
• In recent years, methods have been patented for the use of this strategy
in patient.
Pro-apoptotic Genes
3

• Gendicine is a recombinant adenovirus engineered to express wild-
type p53 (rAd-p53), which is designed to treat patients with tumors
that have mutated p53 genes.
• The process of programmed cell death (apoptosis) is regulated by
cysteine proteases known as caspases.
• The initiator of apoptotic pathway is caspase-9, which is activated
when it is caught by Apaf-1 in presence of dATP and cytochrome-c.
• Caspase-9 has been fused to a CID-binding domain (artificial caspase
activated by chemical inducer of dimerization) and is named
‘inducible caspase-9ʹ (iCaspase-9), which induces the apoptotic
signaling pathway.
Pro-apoptotic Genes
3

A system with a nucleic acid sequence encoding a cell death mediator
protein (CDMP) has been patented.
 The expression of apoptosis promoting activity of the CDMP can be
inducible, for example, by iCaspase-9 to activate the apoptotic
pathway.
Pro-apoptotic Genes
3

There are other apoptotic dysfunctions that make cancer cells resistant
to treatment and induce tumorigenesis.
 Bax, a central cell death regulator, is an indispensable gateway to
mitochondrial dysfunction and a pro-apoptotic member of Bcl-2
family proteins that controls apoptosis in normal and cancer cells.
 Other patented strategies include genes involved in others apoptotic
and cell death mechanisms such as Smac, caspase 3, and TRAIL,
driven by specific promoters.
Pro-apoptotic Genes
3

Strategies to improve suicide gene therapy
Improved vectors for suicide gene delivery
Double-gene therapy of cancer
Combined suicide gene therapy and
classical therapies of cancer
Targeted suicide gene therapy
1
4
3
2

In order to be suitable for clinical applications, a vector must
meet the following requirements:
Low cytotoxicity
Low
immunogenicity
High
transfection
efficiency
Tissue
specificity
Cost-
effectiveness
1
2
3
4
5
1

Suicide gene delivery systems can be divided into three major groups
including:
Viral vectors
Synthetic vectors
Cell-based vectors
A
B
C
1

Viral vectors
 Viral vectors are the most efficient vectors for gene delivery.
 The most common viruses in the field of gene therapy are:
I. Retrovirus
II. Adenovirus (Ads)
III. Lentivirus
IV. Adeno-associated viruses (AAVs)
V. Also, a new vector based on sindbis virus, a blood-borne alpha virus
transmitted through mosquito bites, was patented. It has been shown that
Sindbis virus is able to induce apoptosis in mammalian cells without
carrying out cytotoxic genes.
Viral vectorsA

I) Viral vectorsViral vectorsA Limitations
of viral
vectors
Insertional
mutagenesis
Immunogenicity
1
2

Many efforts were carried out in the development of nanosystems for
gene delivery.
Synthetic vectorsB
These systems efficiently express genes.
They exhibit low-toxicity and low-immunogenicity.
They are safer and cheaper to produce.
Attractive for development into potential clinical applications.

Different non-viral delivery systems have been developed, including:
Synthetic vectorsB
Needle injection
Gene gun
Electroporation
Sonoporation
Magnetofection
Cationic lipids
Polymers
etc.

Cellular therapy based on the use of stem cells is an emerging therapeutic
modality that currently generates great expectations in cancer.
 These cells have many benefits including:
1. Their renewing ability of themselves through cell division, sometimes
after long periods of inactivity.
2. Their potential to differentiate into many cell types in the body during
early life and growth.
3. Their ability to homing to the tumor microenvironment.
Cell-based vectorsC

Mesenchymal stem cells (MSCs) have been used as carriers for in vivo
delivery of various clinically relevant anticancer agents, including
interferon, pro-drugs, or replicative adenovirus.
 The MSCs administered to a subject were able to migrate to the
tumor site.
 These cells may be optimized for use as an off-the-shelf product by
using an immortalized MSC line with immunological characteristics
to forestall a graft-versus-host response.
Cell-based vectorsC

Moreover, the recent patent “Cancer specific suicide gene for cell
based and gene therapy” provides pluripotent and multipotent stem
cells that have been modified to contain an inducible cancer-specific
suicide gene construct that, upon induction, selectively kills stem- or
progenitor-cell-derived cancerous cells.
Cell-based vectorsC

Another invention claimed a method of providing safety of application
of pluripotent stem cells in tissue substituting therapy by means of
artificial chromosomes carrying bicistronic cassette with suicide gene.
 This invention makes possible to:
I. Cancer transformation of transplanted cells to zero risk
II. Development of teratomas
III. Clinical efficiency
Cell-based vectorsC

Double-suicide gene therapy is a promising strategy for the treatment
of advanced cancer.
Co-expression of TK/GCV with CD/5-FC (CD/TK) offers greater
therapeutic efficacy than single-suicide gene therapy and allowed
better outcomes to be achieved with lower prodrug concentrations.
2

The combination of enzyme-activating and prodrug systems has
some drawbacks:
1. Toxic metabolite release.
2. Reduced bioavailability.
For this reason, a new strategy based on double-suicide gene therapy
using gef and apoptin, two killer genes that do not need a prodrug to
be effective in tumor cells, was patented.
The co-expression of both genes enhances the cell growth inhibition
induced by single-suicide gene therapy, taking advantage of the
synergistic anticancer effects of both systems in colon carcinoma cells.
2

 Combining suicide genes with other therapeutic modalities:
i. Promote the therapeutic efficiency and safety
ii. Minimize the side effects of different treatments
 Gene transfer has been proposed as a new strategy to enhance the
efficacy of antitumor drugs in the treatment of intractable or metastatic
cancers.
 The co-administration of chemotherapy and suicide genes has resulted
in enhanced anticancer effects.
Combined suicide gene therapy and
classical therapies of cancer
3

To increase specificity and safety of suicide gene therapy, the expression
of the therapeutic gene needs to be tightly controlled within the target
tissue.
Targeted suicide gene therapy
4
Receptors targets for therapeutic
transgene vectors
Tissue-specific promoters
Disease-specific promoters

Receptors targets for therapeutic transgene vectors
Several cancer-specific receptors may be a molecular marker in the diagnosis
of tumor cells and could be a potential therapeutic target.
In hepatocellular cancer, serotonin receptors 1B & 2B, gamma-aminobutyric acid (GABA) and
gamma-aminobutyric acid A receptor θ subunit and fibroblast growth factor receptor 3 (FGFR3),
are overexpressed.
Claudin clostridium perfringens enterotoxin receptors are upregulated in prostate, breast,
pancreatic, and ovarian cancer cells.
Bcl-2 is overexpressed in breast carcinomas and ovarian carcinomas and the high-affinity laminin
receptor (LAMR) is expressed in several types of human tumors including breast cancer.

Cancer-specific promoters
The main objective of suicide gene therapy is to deliver therapeutic
genes into target cells safely and without harming surrounding healthy
cells.
One possibility is the use of tumor-specific promoters overexpressed
in cancer cells.
They can induce a specific expression of therapeutic genes in the
tumor increasing their localized activity.

There are several cancer-specific promoters employed in gene therapy
such as:
Cancer-specific promoters
Alpha-fetoprotein promoter
Carcinoembryonic antigen promoter
Prostate-specific antigen (PSA) promoter
Human telomerase reverse transcriptase (hTERT) promoter
Human α-lactalbumin promoter
Ovine β-lactoglobulin promoter
Human prolactin-inducible protein-15 (PIP-15) promoter

 The design of viral vectors that allow selective tropism for particular
types of cells and tissues remains a challenge.
To achieve the purpose of targeted killing of tumor cells, in some case
the full potential of Adenovirus gene transfer has not been fully
realized because of the non-specific in vivo tissue distribution of
Adenovirus receptors.
Adenovirus receptors are expressed at low levels in some target
tissues rendering them difficult to infect.
Modification of the tropism

The patent CN 104328140 A solves this problem modifying
adenovirus tropism by integrating the RGD sequence into the
adenovirus cilium zone.
Thus, along with the expression of the bladder epithelium-specific
promoter UPII improved the tropism on the bladder cancer, which
lowly expresses the adenovirus receptor and solves the problem of low
infection ability of the adenovirus vector.

Recently, a next-generation of Adenovirus viral vectors capable of
selective tropism and efficient gene delivery have been developed.
The vector allowed selective killing of U87 glioblastoma cells and
derived xenografts via the HSV-TK, increasing the potency of GCV by
25-fold.

Conclusion
 Although suicide gene therapy has been successfully used in a large
number of in vitro and in vivo studies, its application to cancer patients
has not reached the desirable clinical significance.
 Recent preclinical and clinical studies in cancer models prove the
enormous potential of this strategy when used in combination with
classic therapeutic approaches.
 Thus, this combination therapy can result in enhanced anticancer
effects especially when the different treatments act in diverse
pathways and they would reach all the cell types that make up the
tumor including cancer stem cells (CSCs), making it a new promising
strategy in cancer therapy.

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