2. Introduction
Gene :
Term- Danish botanist
and geneticist Wilhelm
Johannsen(1909)
Greek, gonos: offspring and
procreation
A sequence
of nucleotides in DNA or RNA
that encodes the synthesis of
a gene product, either RNA
or protein
Majority of organisms encode
their genes in long strands
of DNA
3. Introduction
Genome:
Total complement of genes in
an organism or cell stored on
one or more chromosomes
A chromosome consists of a
single, very long DNA helix on
which thousands of genes are
encoded
The region of a particular
gene in its location is
its locus
4. Gene expression
1. Transcription
2. RNA processing
and transfer
3. Translation
Gene expression varies depending on the cell’s function which
results in multiple cell and tissues phenotypes constituting
human body.
5. Cell division
Disorder in cell division-
cell death or proliferation
Phases
G1: enzymes, nucleic
acids
S: DNA
replication/synthesis
G2: cell growth and
duplication
M: replication
G0: resting state
7. Gene therapy
Principle
Can be restorative,
regenerative or
protective in nature
Works by replacing ,
suppressing /
enhancing gene
expression
8. Gene therapy :replacement
Replace the defective genes with normal gene
Targets in rare inherited diseases
Applicability in treatment of inherited disease
Examples :
Functioning Adenosine deaminase replacement in
SCID have effectively reconstituted immune
system
Factor IX gene in Hemophilia B
CFTR gene in cystic fibrosis
10. Gene therapy :enhancement
Can be effective in common acquired diseases- cancers,
arthritis and atherosclerosis
Example : Clinical trial was done for melanoma
Tumor infiltrating cells after surgery, purified, grown and TNF
introduced into the cells
Genetically engineered cells transfused to the patient ,
migrates to the residual tumor site delivering the therapeutic
dose of TNF.
11. Gene therapy: supression
Suppressing key genes
Examples:
RET, protooncogen responsible for medullary carcinoma
thyroid can be suppressed by mutant RET gene
New technique (Nobel prize 2006) : RNA interference(RNAi),
genes are selectively silenced
Off targets effects; but unclear
Human trials on macular degeneration and
neurodegenerative diseases
12. Gene therapy
Disorders under evaluation
Infectious disease trials :HIV
Monogenetic diseases: cystic fibrosis , Hemophilia
B
Polygenetic diseases: rheumatoid arthritis, cancer
13. Gene therapy
In a study : 10 year follow up of SCID subjects out of 20,
18 are still alive including 4 with leukemia and 17 who have
their immunodeficiency corrected
14. Gene therapy: somatic versus
germ cell gene therapy
Somatic gene
therapy
Somatic cells
constituting organs and
postnatal tissues of body
Bone marrow, liver,
tumor cells, muscle,
skin, endothelium,
thyroid
Doesn’t alter the
inherited genetic
material
16. Gene therapy : somatic versus
germ cell therapy
Germ cell therapy
Targets germ cells or those that produce sperm
and ovum
Passed onto a person’s offsprings
Genetic manipulation can alter the genetic
constitution of offspring
Could prevent the inherited diseases
17. Permanent versus temporary
gene therapy
Permanent gene therapy
Requires indefinite
enhancement or blockage
of target gene
Organ resection, cellular
transplantation, growth
hormone deficiency,
juvenile diabetes
Short term efficacy and
long term safety
Temporary gene therapy
Temporary gene regulation
Cancer, cystic fibrosis,
arthritis
Selected beneficial effect
over a limited period of
time
Example in cancer; directly
toxic to cancer cells or
cytokines that initiate
antitumor immune
response.
18. Methods of delivering gene
therapy
1.Direct injection of genetic
material
DNA mediated transfer of
therapeutic genes
Direct delivery of RNA for
gene silencing
2. Viral mediated gene
transfer
Retroviruses
Adenoviruses
Adeno associated virus
3. Non- viral vector mediated
gene transfer
24. Cystic fibrosis (CF)
Single mutation and autosomal recessive
inheritance
Mutation involving CF gene in long arm of
chromosome 7
CF gene encodes a protein- cystic fibrosis
transmembrane conductance regulator(CFTR
protein)
Commonest lethal genetic disorder involving the
wealthiest racial groups, so research funding never
a problem
26. Nasal model of gene therapy
Promising in cystic fibrosis
Nasal manifestations
Gene administration by virus vectors safer via nasal
cavity
Nasal and sinus mucosa have fairly large surface area,
adequate for absorption of the vector
Adenovirus commonly used, has propensity to adsorb
to nasal mucosa
Treatment assured by measuring potential difference
across the nasal mucosa and easy to perform
28. Pulmonary model of gene
therapy
Life threatening complication of cystic fibrosis due
to effect on lungs
Adenovirus easily adheres to the alveolar mucosa
Has deleterious effects on the lung tissue causing
death, thus cautions to be made while using the
model
29. Adenovirus vectors
Have a tropism for respiratory mucosa
Recombinant virus is prepared by replacing DNA sequence
responsible for replication of virus with that of DNA sequence
responsible for secretion of CFTR protein deficient in CF
patients
Human respiratory mucosa has sufficient immunity to prevent
adenovirus infections , hence administration via pulmonary
route may not be effective
Nasal route ideally suited in cystic fibrosis.
31. Adenovirus gene therapy; role of
calcium chelators
Vectors are aimed at
epithelial surfaces
In pulmonary epithelium,
viral receptors are located
in basolateral membrane
away from the surface
Use of calcium chelators
will cause transient
disruption of tight epithelial
junctions allowing vector
access to the basolateral
membrane
32. Adeno associated virus
Serotypes 5 and 6 enters airway cells from the apical surface
Least toxicity and minimal immuno-reaction
Potential integration into host genome
Direct administration into maxillary sinuses attempted, has
reasonable success and do not cause sinusitis
Measurement of maxillary sinus voltage helps in
determination of therapeutic end point
33. Lentivirus
Feline immunodeficiency
virus
Can integrate and persist in
the host genome
Can transduce into non
dividing cells
Helpful in the pulmonary
mucosa whose turnover is
rather low
34. Non viral vectors
Purified /naked DNA in
plasmid form
Gene gun/ballistic delivery
system-on exposed
surfaces
Liposomes : DNA coated
liposomes gets
incorporated into the cell by
endocytosis or fusion
Non immunogenic
Gene transfer inefficient
35. Gene therapy in head and neck
cancer
Increased appreciation of biological basis of oncogenesis
Understanding of gene targets to potentially correct by either
replacement or blockage of the effect of mutated gene
Therapy strategies:
Immune modulation
Restorative gene replacement
Selective oncolysis
chemosensitization
36. Ideal gene therapy system
To achieve expression of gene of interest in the targeted
cancer cell
Malignant cells need to be targeted
Binding and internalisation of genes by the targeted cells
Gene should escape the endosomal degradation and reach
the nucleus
Nuclear expression-once inside the nucleus the quantity of
gene expression should be adequate and stable
37. Chemosensitization
Selective sensitization of cancer cells to chemo/radiotherapy
Ideal way to kill cancer cells
Target only malignant cells
Herpes simplex thymidine kinase can be delivered to cancer
cells making them more susceptible to Gancyclovir
38. Chemosensitization
Bystander effect ensures that the cancer cells spread these
genes into cells surrounding them via cell to cell contact
Transfer of p53 gene sensitizes the cancer cell to undergo
apoptosis following chemo/radiotherapy
39. Immune modulation
Immune dysfunction at the site of tumor causes malignant
cells to thrive
Immunological ignorance/down regulation of major
histocompatibility complexes contributes for the modulation
Over expression of down regulated cytokines, proven to be
toxic due to capillary leak syndrome
40. Restorative gene therapy
Mutations of p53 and p16 genes are common in squamous
cell carcinoma of head and neck thus restoration of these
genes could enhance apoptosis of tumor cells
Can inactivate proto-oncogene production
Repair of deranged apoptotic pathway in tumor cells could
help in controlling malignancy
41. Selective oncolytic viruses
Infection with wild type adenovirus can cause excessive
replication of the viruses leading on to cell death
If these viruses could be harnessed to replicate inside cancer
cells alone then preferential targeted destruction of tumor
cells can occur
ONYX-015, adenovirus replicate inside cancer cells that lack
functional p53 gene
If administered intravenously can take care of distant
metastasis.
42. Gene therapy in head and neck
cancer
TP53, most commonly mutated
tumor suppressor gene, has been
the focus of some prior
investigations into gene therapy in
HNSCC.
In China, recombinant p53
protein delivered via an adenoviral
vector (Gendicine) is in phase II/III
clinical trials (Liu et al., 2013) as
an adjuvant therapy to standard of
care (surgery, chemotherapy and
radiation).
In the United States,
investigations of same remain in
early clinical phases (Yoo et al.,
2009).
Investigation into restoration of other tumor
suppressor genes in HNSCC is lacking.
43. Newer field for gene therapy
1. Plastic and reconstructive surgery
Reconstructive tissue flaps and wound healing
Repair and regeneration of irradiated tissues
2. Laryngology
3. Otology and neurotology (adenoviruses and
herpesvirus derived vectors)
45. Newer field for gene therapy
Columbia University, Department of Otolaryngology,
Head and Neck Surgery
46. Why GENE therapy ?
New therapeutic approach
Site-specific gene expression
Improved efficacy and safety
Improved routes of administration and compliance
Preventive medicine and a reduction in health care costs
47. References
1. Scott brown’s Otorhinolaryngology Head and Neck Surgery volume 1, 8th
edition
2. Scott brown’s Otorhinolaryngology Head and Neck Surgery volume 1, 7th
edition
3. Cummings otolaryngology volume 2, 6th edition
4. The potential for tumor suppressor gene therapy in head and neck cancer
Birkeland AC1, Ludwig ML1 , Spector ME1, Brenner JC1.
5. A Study of the Gene Therapy CGF166 in Patients with Severe-to-Profound
Hearing Loss, Columbia University, Department of Otorhinolaryngology
Head and Neck Surgery.
