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G M Institute of Technology

Department Of Biotechnology,

Presented by,
Avinash V
• Introduction
• Gene therapy overview
• Suicide gene therapy
• In vitro and In vivo mechanism
• Advantages
• Limitations
• Future
• Conclusion
Suicide – to kill oneself
Birth of Gene therapy approaches –
frequency of occurrence , lack of
efficacy of available oncogenetic
treatment, diverse genetic
background of different malignant
diseases
Suicide gene therapy
 Genetic manipulation
 Gene Therapy- original concept
 70’s – utility of DNA as Theurapetic agent
 1971 – First unauthorized gene transfer into
humans by stanfield Rogers
 80’s –various preliminary studies
 1990 (s) – first approved gene treatment
treatment for trial, better and safer vector
development, increase in understanding of many
diseases
 2000 – First patients cured with aid of gene
therapy
 2003 – first gene therapy protocol approved for
clinical practice in china against head and neck
squamous cell carcinoma
Target disease Gene delivered Phase of
Clinical
Inherited disorders development
Hemophelia FIX or FVIII I
Cystic fibrosis CFTR I
Chronic granulomatous p47phox I
disease
Acquired diseases
Cancer
head and neck squamous cell p53 Approved
carcinoma
Glioma HSV-TK I/II
Alzheimer’s disease NGF I
Lower limb ischemia VEGF II
 Features of optimal vectors – Ability to
transduce cells of different tissue, target the
vectors to certain tissue, And a stable,
sufficiently long lasting and regulated
transgene expression in target tissue.
 Problems – side effects due to hazardous
interaction and immunological reactions
 Viral vectors – adeno, retro, lenti, herpes
viruses
 Non viral vectors – less toxic, low
immunogenicity , low efficacy, unlimited
transgene capacity. – cationic liposome's,
cellular gene delivery, stem cells
66.4 percent gene therapies are
aimed against cancer
Focus on 3 major themes – discover
new means of killing, improvement of
gene delivery and vector systems,
translation of preclinical studies to
clinical protocols and trials
Melanoma, leukemia, prostrate,
ovary, squamous cell carcinoma
Gene therapy strategy Example gene
p53
Tumor suppressor gene BRCA1
RB
ERBB2
Oncogene
KRAS
Anti-angiogenesis VEGF

Immunotherapy IL-2

Chemo-protective therapy 1

Adeno virus
Virotherapy, oncolysis
Herpes virus
HSV-tk
Suicide gene therapy
CD
Original concept
Basic strategy
herpes simplex virus thymidine
kinase (HSV-tk) gene coupled with
the pro-drug ganciclovir (GCV)
cytosine deaminase (CD) gene
coupled with the pro-drug 5'
fluorouracil (5-FU)
xanthine guanine phosphoribosyl
transferase (XGPRT)
purine nucleoside phosphorylase
(Besnard et al., 1987, Mroz and
Moolten., 1993).
Thymidine kinase
HSV-tk – ability to phosphorylate
broad range of guanosine analogues
like Ganciclovir, acylovir, buciclovir,
penciclovor
Molten observed
Culver and coworkers – first noticed
GCV-p from HSV-tk positive cells to
wild type
Touraine and co-workers – relation
b/w gap junctions and Bystanders
effect
Other routes also proposed by many
other researchers
Initial
suggestions from studies
Bi and coworkers (1993)- used
radiolabelled GCV
Gap junctions
In vivo Mechanism
rapidly replicating tumor cells are
more susceptible to impairment of
DNA synthesis
chemotherapy resistant tumors can
be made sensitive when genetically
modified
HSV-tk/GCV-treated tumor cells have
the ability to kill neighboring tumor
cells through the bystander effect
 showed enhanced growth, invasiveness,
resistance to chemotherapy of tumors transduced
with HSV-TK.
 GCV uptake and its low affinity to HSV-TK may
also limit the clinical efficacy of this treatment
form
 GCV might not be the best substrate for HSV-TK
due to its inadequate transport into the cells as
well as the low levels of GCV phosphorylation.
 It has also been noticed that sensitivity to GCV
and the Bystanders effect varies between
different tumor cells lines,
 Showed that GCV uptake increased along with the
percentage of HSV-TK expressing cells, which was
 combine traditional cancer treatment methods with
gene therapy.
 Enhanced therapeutic effect has also been observed
by combining prodrug therapies.
 combining two widely used suicide genes
 HSV-TK in combination with other genes has
demonstrated increased efficacy
 Immune system related gene has been combined with
HSV-TK
 In addition to the combinations of different genes and
HSV-TK/GCV therapy, there are a number of other
interesting treatment combinations.

promising solution to kill tumors


 Even though with all these limitations and hurdles that this
suicide gene therapy is facing, there seems to be a lot of
research and scientific works going on to improve the
efficiency and effectiveness of this way of treating cancer.
May be in a few years the hurdles will be overcome and the
suicide gene therapy would come to the markets as the
most efficient and effective therapy for Cancer and other
tumors.
 TIINA WAHLFORS: Enhancement of HSV-TK/GCV suicide gene therapy
of cancer
 Tumor killing using the HSV-tk suicide gene
 Rajagopal Ramesh1, Aizen J. Marrogi1 and Scott M. Freeman2,3
 1. Department of Surgery and Gene Therapy Program, LSU School of
Medicine, New Orleans, Louisiana, USA.
 2. Department of Pathology, Tulane University School of Medicine, New
Orleans, Louisiana, USA.
 Pasanen T., Karppinen A., Alhonen L., Jnne J. and Wahlfors J. Polyamine
biosynthesis inhibition enhances HSV-1 thymidine kinase/ganciclovir-
mediated cytotoxicity in tumor cells. Int J Cancer (2003) 104, 380-388
 Pasanen T., Hakkarainen T., Timonen P., Parkkinen J., Tenhunen A.,
Loimas S. and Wahlfors J. TK-GFP fusion gene virus vectors as tools for
studying the features of HSV- TK/ganciclovir cancer gene therapy in
vivo. Int J Mol Med (2003) 12, 525-531
 Wahlfors T., Hakkarainen T., Jnne J., Alhonen L., and Wahlfors J. In vivo
enhancement of Herpes simplex virus thymidine kinase/ganciclovir
cancer gene therapy with polyamine biosynthesis inhibition. Int J
Cancer, in press.
 Wahlfors T., Karppinen A., J?nne J., Alhonen L. and Wahlfors J.
Polyamine depletion and cell cycle manipulation in combination with
HSV thymidine kinase/ganciclovir cancer gene therapy. Int J Oncol, in