Cancer Gene Therapy- Developments and Future Perspectives
- SourceAvailable from: Dr. Mohammad Abdullah Yusuf
Dataset: Gene therapy: Where do we stand?[Show abstract] [Hide abstract]
ABSTRACT: The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. 'Gene Therapy' covers both the research and clinical applications of the new genetic therapy techniques currently being developed. The application of molecular biology has revolutionized researchers understanding of many diseases and has been readily applied for diagnostic purposes. Now-a-day this is originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions, including those adversely affecting a patient's quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. It is not very far, the justifiable optimism that with increased biotechnological improvement, gene therapy will become a standard part of clinical practice.
Article: Gene therapy: Where do we stand?[Show abstract] [Hide abstract]
ABSTRACT: The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. 'Gene Therapy' covers both the research and clinical applications of the new genetic therapy techniques currently being developed. The application of molecular biology has revolutionized researchers understanding of many diseases and has been readily applied for diagnostic purposes. Now-a-day this is originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions, including those adversely affecting a patient's quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. It is not very far, the justifiable optimism that with increased biotechnological improvement, gene therapy will become a standard part of clinical practice.IOSR Journal of Pharmacy and Biological Sciences. 05/2013; 6(4):31-34.
Cancer Gene Therapy - Developments
and Future Perspectives
David A Good1,2, Wei Duan3, Jozef Anné4 and Ming Q Wei1
1Division of Molecular Medicine and Gene Therapy, Griffith Health Institute, ?
Schoolof Medical Science, Gold Coast campus, Griffith University, Qld, ?
2School of Physiotherapy, Australian Catholic University, ?
McAuley Campus, Banyo, Qld,?
3School of Medicine, Deakin University, Waurn Ponds, VIC., ?
4Rega Institute for Medical Research, ?
Minderbroedersstraat 10, Leuven, ?
Cancer is the second leading cause of death in the Western world. Whilst there are many
therapies which can significantly improve patient outcomes, there is still no definitive
“cure” for cancer. Over the last decade we have witnessed gene therapy developing at a
very fast pace representing a potential new and more effective modality for the treatment of
cancer. By the end of 2010, over 1060 gene therapy protocols have been proposed or trialled
in the clinical setting for various cancers; this figure represents over 64% of all gene therapy
trials in humans in the United States. From many of these trials modest therapeutic
responses have been reported, however, unequivocal proof of clinical efficacy is still to be
seen. In 2003, the first commercially produced gene based product was manufactured by
Shenzhen SiBiono GeneTech. Gendicine™, a replication-incompetent recombinant human
Ad5-p53, was approved by the Chinese State Food and Drug Administration to treat head
and neck squamous cell carcinoma. Again in 2006, Shanghai Sunway Biotech, commercially
released a conditionally replicative adenovirus therapy, Oncorine™. However, it is fair
to say that cancer gene therapy has yet to realise its full potential.
2. The gene therapy vector systems
An important feature for any successful gene therapy protocol is the vector system. To date,
a number of vectors have been developed, including both viral and non-viral based therapy
systems (Table 1). Each of these vectors possesses a number of unique features and each has
its own advantages and disadvantages. The most promising existing vectors are the
replication-competent oncolytic viral vector based gene therapy systems, particularly the
Gene Therapy - Developments and Future Perspectives
Easy to produce, Can achieve
high titre, High efficiency of gene
Relatively easy to produce, Has
ability for gene integration
Can be made through packaging
cells, Has ability for gene
Has Neurotropic affinity, Can be
applied to Neurons or Glioma
Easy production, less
Simple, easy to make and easy to
Large DNA virus, many viral
genes, often Immunogenic
Semliki Forest virus
Wide type Adenovirus
Low efficiency of gene
transfer, dividing cells only
Large DNA virus, many viral
Difficult to purify
Table 1. List of commonly used vectors for cancer gene therapy as well as their advantages
3. Viral vector systems
3.1 Adenoviral vectors
Adenovirus vector (AV) is the most commonly studied and most widely used system in
cancer gene therapy. It is of particular use for cancer gene therapy applications, where
temporary gene expression is acceptable or even beneficial. There are several serotypes, but
the currently employed AVs in clinical trials are mostly based on serotype 5. These vectors
can replicate highly and have demonstrated efficient gene transfer into various types of
cancer cells. Two AVs related gene therapy products have been approved for clinical use
in patients suffering from head and neck cancers in China, replication-incompetent
recombinant human Ad5-p53, and the other is a conditionally replicative adenovirus
Several other adenoviruses, based on canine, porcine, bovine, ovine and avian adenoviruses
have been developed. The ovine AV is based on serotype 7 and was developed in Australia.
Preclinical testing of ovine AV on prostate cancer in animal models has shown therapeutic
Unfortunately, AVs contain many viral genes which encode for major proteins that elicit a
strong host immune response. Of particular concern is the release of cytotoxic T
lymphocytes that lyse cells expressing the recombinant genes. Newer generations of AV
vector have been designed to overcome some of these problems and initial results are
encouraging. New techniques involved in removing the recombinant viral genes and
transfecting the non-recombinant plasmid with a helper virus and then separating the
helper virus with sedimentation techniques have been developed. Improvements in helper
virus have also been trialled that reduces "floxed" helper virus production 1000-fold, but this
method still has a 1% wide type (WT) contamination thus still allowing the possibility of in
vivo recombination. With regard to AV-mediated cancer treatment, high-level tumour
transduction remains a key developmental hurdle. To this end, AV vectors possessing
infectivity enhancement and targeting capabilities should be evaluated in the most stringent
Cancer Gene Therapy - Developments and Future Perspectives
model systems possible. Advanced AV-based vectors with imaging, targeting and
therapeutic capabilities have yet to be fully realized; however, the feasibilities leading to this
accomplishment are within close reach.
3.2 Adeno-associated virus (AAV)-based vectors
AAV-based vectors have been shown to be non-toxic and undergo widespread cellular
uptake in preclinical evaluation. A recent study has compared five different AAV strains
and amongst them, serotype 2 was proven to be the most efficient killers of tumour cells. In
another study, serotype 8 AAV vector encoding a soluble vascular endothelial growth factor
(VEGF) receptor was able to halt tumour growth in several rodent glioma models. However,
difficulties in the development of packaging cell lines for AAV, as well as bulk production
and vector purification have been reported as problematic. A new system was
developed recently to scale up and bulk production of AAV from insect cells which may
solve some of these existing problems.
3.3 Retroviral vectors
The earliest system that was developed for gene therapy was the murine Moloney
leukaemia virus (MoMLV)-based retroviral vector. More than 23% of all gene therapy trials
in patients for various diseases have used a replication-defective or competent MoMLV
vector system. This vector’s unique ability to transduce dividing cells makes it an ideal
choice of vector for continuously dividing and fast growing tumours, such as gliomas.
However, poor vector penetration and lack of vector migration away from the injection site
is usually seen. To overcome this effect, replication competent MoMLV was developed.
A 2005 publication showed complete transduction of human U87 glioma xenografts in nude
mice after a single intracranial (i.c.) injection of replication-competent MoMLV. In this
study, viral envelope was stained positively in glioma cells away from the injection sites.
Most importantly, no virus was detected in any non-tumour tissue which is good indication
of strict tumour specificity.
Potential limitations of retroviral vector systems are related to their ability to activate
cellular oncogenes and inactivate tumour suppressor genes by insertional mutagenesis. One
study used MoMLV to transduce bone marrow stem cells for the therapy of severe
combined immune deficiency syndrome (SCID), following treatment 4 out of 11 children
subsequently developed leukaemia. In addition to improving safety, there are also
studies that have shown that dissemination of the vectors in solid tumours still needed to be
improved in order to reach clinical efficacy.
Lentiviral vectors are a more recently developed and complicated retroviral vector, based on
the Human or Bovine Immunodeficiency Virus. They have all the unique features of
MoMLV and have been shown to transduce post mitotic cells in vitro and in vivo. Studies
with the Human Immunodeficiency Virus (HIV)-based vectors have shown efficient gene
transfer in tumour models. An understandable reluctance in the use of potentially
pathogenic HIV vectors in humans has been seen, although a clinical trial assessing use of
lentiviral vector for the therapy of AIDS is underway. To reduce the risk of
seroconversion in human patients several bovine based vector systems have been
developed, which have the advantage of less or no pathogenicity in humans. We have also
developed a bovine lentiviral vector system based on the Jembrana Disease virus (JDV)[23,
24]. JDV only causes disease in a specific species of cattle in the Jembrana district in Bali,
Gene Therapy - Developments and Future Perspectives
Indonesia, but does not affect humans. Pathological changes in cattle include intense non
follicular lympho-proliferation by reticulum and lymphoblastoid cells in lymphoid organs.
Protein and genome sequence studies have confirmed that JDV has a genome of 7732
nucleotides and structure and organisation similar to other members of the lentivirus
family. More importantly, JDV possesses several features in common with HIV that are very
attractive as a vector, including a high replication rate and the ability to efficiently integrate
into chromosomes of non-dividing and terminally differentiated cells.
3.4 Herpesvirus-based vectors
Vectors based on herpesviruses are well-developed and have progressed to clinical trials. As
with other viral vectors, replication-defective vectors have not shown much potential. The
first replication competent vector was based on a mutant strain, where the vectors are
deleted from the main neurovirulence gene r34.5, thus restricting its ability to replicate in
adult central nervous system and to form latency. However, later study showed that the
mutant strain which had the deletion of the r34.5 gene also had reduced capacity for
replication inside tumour cells. New vectors were developed with a deletion of the
ICP47 gene which does not appear to impact on efficient replication.
Pre-existing immunity may pose a problem that limits the clinical efficacy of herpesvirus-
based vectors. This immunity prevents the transduction of peripheral organs and also can
cause liver toxicity. However, a recent mutant strain-secreting cytokine granule macrophage
colony stimulatory factor (GM-CSF) or IL-12 was shown to be effective in liver cancer
therapy in a murine model which likely involves both direct viral oncolysis and actions of
specific immune effector cells.
3.5 Viral replicons and transposons
Semliki Forest virus (SFV) subgenomic replicons (i.e. non toxic replication) have been
developed that allow stable expression of a required gene e.g. beta-galactosidase (beta-Gal)
in mammalian cell lines. Studies showed that expression remained high (approximately 150
pg per cell) throughout cell passages.
Since construction of the Sleeping Beauty transposon from defective copies of a Tc1/mariner
fish element, new vertebrate genetic manipulation tools (i.e. transposase enzymes) have
become available for gene therapy. This particular transposase in the system binds to the
inverted repeats of salmonid transposons that surround the insertion gene and mediate
precise 'cut and paste' into fish, mouse and human chromosomes. Potential problems with
the use of transposons for gene therapy may arise from having no 'off' switch for the
transposase and the relatively low quantities of integrated product, either of which would
make retroviral intergrase a more suitable or
alternative enzyme for chromosomal
3.6 Targeted viral vectors
While efforts have been focused on the continuing refinement of various vector systems,
several obstacles remain, primarily the low efficiency of gene delivery into target tumour
cells. The vascular endothelial wall is a significant physical barrier prohibiting access of
systemically administered vectors to the tumour cell. To overcome this obstacle, strategies
are currently being developed to take advantage of transcytosis pathways through the
endothelium. An AV vector targeted to the transcytosing transferrin receptor pathway,
Cancer Gene Therapy - Developments and Future Perspectives
using the bifunctional adapter molecule has been constructed. The transcytosed AV
virions retain the ability to infect cells, establishing the feasibility of this approach. However,
efficiency of AV trafficking via this pathway is poor. Other efforts are directed towards
exploring other transcytosing pathways such as the melanotransferrin pathway, the poly-
IgA receptor pathway, or caveolae-mediated transcytosis pathways. There are hopes to
develop mosaic AV vectors incorporating both targeting ligands directed to such
transcytosis pathways as well as ligands mediating subsequent targeting and infection of
tumour cells present beyond the vascular wall.
3.7 Viral vector-associated multifunctional particles (MFPs)
Nanotechnology has recently been incorporated into viral vector systems in the form of
multifunctional particles (MFPs). Nanotechnology is defined as the development of devices
of 100 nm or smaller, having unique properties due to their scale. The devices that are being
developed generally incorporate inorganic or biological material. In this regard, the
coupling of inorganic nano-scale materials to targeted AV vectors has much potential for
tumour targeting, imaging and amplified tumour killing capacities. For example, magnetic
nano-particles have recently received much attention due to their potential application in
clinical cancer treatment; targeted drug delivery and magnetic resonance imaging (MRI)
contrast agents. However, despite the useful functionalities that might derive from metal
nanoparticle systems, the lack of targeting strategies has limited their application to
locoregional disease. Thus, tumour-selective delivery is the key to improve therapeutic
applications of this technology.
AAV has been developed with MFP, by virtue of genetic capsid modifications, to
incorporate additional functionalities, such as modified fibres, combined with imaging
motifs on the pIX protein, to simultaneously target tumour cells while monitoring viral
replication and spread. Herpes simplex virus thymidine kinase (HSV TK) has been
incorporated at pIX site of the AAV capsid. This enzyme is compatible with available PET
imaging ligands such as 18F-penciclovir, providing an imaging system for viral replication
that can directly be translated for clinical applications. Interestingly, HSV TK is an enzyme
that has utility in so-called suicide gene therapy, in which the expressed enzyme converts a
substrate such as ganciclovir to its phosphorylated metabolite, which can then be further
phosphorylated by cellular kinases to a toxic metabolite, causing cell death. Also,
tumour cells expressing this gene product induce the death of adjacent cells via the so-called
'bystander effect', thus representing an 'amplifying strategy' as mentioned above.
3.8 Oncolytic virus
An oncolytic virus is a virus that has the ability to infect cancer cells and cause oncolysis. It
is with obvious reasons that these types of viruses have received much attention in the field
of cancer therapy as they can result in direct destruction of the tumour cells. Initial research
into the anticancer potential of oncolytic viruses examined naturally occurring oncolytic
viruses including adenovirus, poliovirus and Coxsackie virus[33-36]. However, these studies
highlighted a number of limitations with naturally occurring oncolytic viruses including
uncontrolled infection, incomplete oncolysis and the development of an immune
response[33, 35]. The stimulation of the immune system prevented the virus from
destroying the cancer and therefore reduced the efficacy of the treatment. Due to these
limitations many researchers discontinued their research into naturally occurring oncolytic