Gene therapy

gene

1. Dr. SANJAY MAHARJAN PG resident, ENT-HNS MTH, Pokhara “ Gene therapy in head and neck cancers”

2. HISTORY: 1963 - Id ea of gene therapy was introduced by Joshua Lederberg 1980s - Gained momentum 1990 - First FDA-approved successful gene therapy treatment of X- linked SCID 1999 - Death of Jesse Gelsinger in a gene-therapy experiment 2006 - Scientists at National Institutes of Health (Bethesda, Maryland ) successfully treated metastatic melanoma in two patients

3. 2011 - Medical community accepted that it can cure HIV as in 2008 , Gero Hutter has cured a man from HIV using gene therapy

4. INTRODUCTION: HNSCC: Malignant tumours of squamous cell origin arising from mucosal surfaces of upper aerodigestive tract, salivary glands, paranasal sinuses, and skin of head and neck Mainstay of treatment for HNSCC is surgery or radiotherapy, +/- chemotherapy Results in 60% 5 year survival This figure has remained largely unchanged for 30 years

5. Gene therapy is “ the deliberate introduction of genetic material into patient's cells in order to treat or prevent a disease ” Loco‐regional nature of HNSCC makes it accessible for both intratumoral injection and tissue biopsy

6. GENETIC CHANGES IN CANCER Normal cell cycle is regulated by numerous genes; proto‐oncogenes and tumour suppressor genes, held in equilibrium Increased (proto‐) oncogene or reduction in tumour suppressor gene expression  aberrant proliferation, hence “cancer” Hallmark changes of a cancerous cell

7. Cancer gene therapy is based on insertion of a gene (transfection) into a cell This new DNA is then “transcribed” to make mRNA which encodes a specific protein that is made through translation

8. TYPE OF GENE THERAPY Corrective Cytoreductive Immunomodulatory

9. CORRECTIVE GENE THERAPY Attempts to block oncogenes or replace tumour suppressor genes Tumour suppressor gene in HNSCC and most other forms of cancer is p53 Damage to genetic material within cell  protein encoded by p53 gene stops cell cycle by binding to DNA If damage is not repairable  triggers cell death (apoptosis ) Alteration to p53 results in continued propagation of damaged cell line

10. Gendicine from Schenzhen SiBono GenTech , China. commercially available gene therapy agent for HNSCC based on p53 135 HNSCC pts (77% stage III or IV) were randomised to receive radiotherapy alone or in combination with Gendicine Replacement of p53 results in reduced HNSCC growth and increased radiochemo ‐sensitivity

11. Effects of oncogene abnormalities can be overcome by blocking the faulty gene May be by : Inserting DNA into cell which binds & blocks oncogene expression (e.g. Transfecting antisense cdna or oligonucleotides) Inhibiting oncogenes' DNA from making RNA (transcription) and/or RNA from making protein (translation) No examples to date for HNSCC

12. CYTOREDUCTIVE GENE THERAPY Aims to directly or indirectly kill the cancerous cell Can be done by Augmenting effects of other anti‐cancer therapies; chemotherapy Concentrating cytotoxic agents in cancerous cells Interfering with tumor's blood supply or Inducing apoptosis

13. Augmentation of chemotherapy: By either a drug sensitisation or resistance approach Sensitisation approach: Gene is transfected to convert a pro‐drug into its active metabolite Allows drug conversion and a high level of active drug only in tumour bed e.g. Herpes simplex virus thymidine kinase (TK) gene, which converts gancyclovir into its cytotoxic triphosphate

14. Resistance approach: Drug resistant gene is added into normal cells sensitive to chemotherapy, so that they can resist chemotherapy Allows higher doses of chemotherapy to be used

15. Concentrating radionucleotides : Gene encoding membrane protein responsible for uptake of iodide is sodium iodide symporter This gene can be inserted into other cancer cells to cause them to concentrate radioisotopes of iodine Can be used for imaging and to administer concentrated local dose of radiotherapy

16. Anti‐ angiogenic : Targeting new blood vessel formation By up regulating anti‐ angiogenic or down regulating pro‐ angiogenic factors Pro‐apoptotic: Normal cells are programmed to kill themselves and is under numerous controls such as tumour necrosis factor (TNF) These control mechanisms can be targeted Yet to reach any clinical trials

17. IMMUNOMODULATORY GENE THERAPY: Modification of immune response to cancer By introducing gene into cancer cells which produces foreign protein on cell's surface This tumour specific antigen allows cell to be seen and destroyed by immune system Cytokines or immune regulatory proteins can be introduced Cytokine gene transfer can be performed in vivo or ex vivo

18. MONITORING OF GENE THERAPY: Indirectly through cross‐sectional imaging Excised and examined by immuno-histochemical methods Molecular imaging to monitor gene therapy By introducing a “reporter gene” Based upon the premise that cells with transfected gene concentrate or activate a marker

19. GENE DELIVERY (VECTORS): Main limiting factor to gene therapy is accuracy and efficacy of delivery of gene by gene delivery vector Route of delivery almost always direct tumour injection Ideal vector: Highly specific (targeting only tumour cells) Highly efficient (all targeted cells become transfected) Safe Unfortunately, so far this ideal does not exist

20. Characterized as Viral vectors Non‐viral vectors Non‐viral vectors: Physically forcing DNA into cell by direct injection in tumor

21. Methods: Electroportation  electric current increases cell permeability

22. Bio‐ballistics (gene gun)  gold particles coated with DNA “shot” into superficial tissue; ultrasound increases permeability of cell membrane

23. High pressure hydrodynamics Uses hyrdrodynamic pressure to penetrate cell membrane Rapid, high volume DNA solution injection  increased permeability of capillary endothelium; forms pores in plasma membrane

24. Also possible with chemical carriers such as liposomes carrying cationic lipids and polymers Further enhanced by anionic ph sensitive peptides Advantage: Less immunogenic and hence may be given repeatedly Can carry more DNA Cheaper to produce Disadvantage: Low transfection rates

25. Viral vectors: Viruses rely on transcriptional apparatus of eukaryotic cell for replication Pathogenic elements of viral genome are removed Replaced by exogenous genes with or without added specificity for infection of cancer cells Virus itself can also exert an anti‐cancer effect— “ oncolytic viruses”

26. Types of viral vector: According to whether their genome integrates into host cell DNA (retroviruses and lentiviruses ) Or persists in cell nucleus as episomes (adenovirus, adeno ‐associated viruses ( aavs , herpes virus)) According to virtue of their ability to replicate ( oncolytic ) or replication deficient Most commonly used vectors in research

27. Retroviruses: Mainly used as ex vivo Target cells are removed from pt  genetically modified  reimplanted Have a natural tendency to transduce dividing cells Risks: Retroviral infection May disrupt host genome ( insertional mutagenesis)

28. Lentiviruses : Members of retrovirus Ability to infect non‐dividing cells

29. Adenoviruses: Strongly immunogenic Can be either replication defective or replication competent Replication defective: Can be produced in large amounts in producer cell lines Ability to infect non‐dividing cells Not inserted into host genome (minimal risk of insertional mutagenesis)

30. Herpes simplex viruses: Non‐replicating herpes simplex virus (HSV‐1 ): Has ability to persist after initial infection in a latent state in neuronal cells for lifespan of cell Have large cloning capacity - allows for simultaneous delivery of several genes No benefit in HNSCC therapy so far

31. Replicating viral vectors: Destruction of cell  new genetic material is also destroyed In order to be successful the effect of gene therapy must be able to spread to surrounding cells Can be done by replication competent vectors Require limited initial transduction of target cells

32. Replication competent Adeno virus: Most commonly studied oncolytic viral vectors One such is ONYX‐015 Has gene responsible for binding to and inactivating p53 removed cell Resulting in a virus unable to replicate in normal cells but capable of replicating in p53 negative cells

33. Phase II trials of 40 patients with recurrent HNSCC No viral replication or toxic effects in normal tissue Tumour regression in 10% Tumour growth stabilisation in 62% Disease progression in 29% In earlier stage HNSCC in conjunction with cisplatin and 5‐fluorouracil (5‐fu)  response rate of 63% versus expected 35% was observed

34. Replicating herpes simplex viruses: With deletion of genes from HSV which control virulence (e.g. ICP6 and/or ICP34.5) virus depends on dividing host cells to replicate  results in cancer cell selectivity Oncovex : Oncolytic HSV with both these deletions and added GM‐CSF

35. Other replicating viral vectors Newcastle virus: Replicates in cells with defects in interferon signalling pathways Oncolytic strain termed P701 , administered intravenously, has undergone phase I trials in 79 patients 22 % of patients tumours stopped growing

36. Vaccinia virus: By deleting thymidine kinase (TK) gene they can only replicate at certain phases of cell cycle and in cancer cells Ability to carry large quantities of DNA, therefore multiple genes Long history of their safety in clinical use