Suicide Gene Therapy

G M Institute of Technology Department Of Biotechnology,

Presented by, Avinash V

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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 development Inherited disorders Hemophelia

FIX or FVIII

I

CFTR

I

p47phox

I

p53

Approved

HSV-TK

I/II

Alzheimer’s disease

NGF

I

Lower limb ischemia

VEGF

II

Cystic fibrosis Chronic granulomatous disease

Acquired diseases Cancer head and neck squamous cell carcinoma Glioma

 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

Oncogene

ERBB2 KRAS

Anti-angiogenesis

VEGF

Immunotherapy

IL-2

Chemo-protective therapy

1

Virotherapy, oncolysis

Suicide gene therapy

Adeno virus Herpes virus HSV-tk 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 

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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

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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.

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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/ganciclovirmediated 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