gene

1. Dr. SANJAY MAHARJAN
PG resident, ENT-HNS
MTH, Pokhara
“GENE THERAPY IN
HEAD AND NECK
CANCERS”

2. HISTORY:
• 1963 - Idea 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
1. Corrective
2. Cytoreductive
3. 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
Gene +
Radiotherapy
93% response
rate
64%
complete
remission
Radiotherapy
alone
79% response
rate
19% complete
remission

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

19. MONITORING OF GENE THERAPY:
1. Indirectly through cross‐sectional imaging
2. Excised and examined by immuno-histochemical methods
3. 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

20. 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

21. • Characterized as
1. Viral vectors
2. Non‐viral vectors
•Non‐viral vectors:
Physically forcing DNA into cell by direct injection in tumor

22. • Methods:
Electroportation  electric current increases cell
permeability

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

24. 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

25. 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

26. •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”

27. • 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

28. •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)

30. •Lentiviruses:
Members of retrovirus
Ability to infect non‐dividing cells

31. •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)

33. •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

34. •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

35. • 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

36. • 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

37. • 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

38. •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

39. •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