gene therapy is an advanced form of treatment for high grade glial tumor

1. GENE THERAPY
DR AMIT KUMAR

2. • Gene therapy is the process by which nucleic
acids are transferred into cells to generate a
therapeutic effect

3. First Approved GeneTherapy
• On September 14, 1990 at the U.S. National
Institutes of Health, W. French Anderson M.D.
and his colleagues R. Michael Blaese, M.D.,
C. Bouzaid, M.D., and Kenneth Culver, M.D.,
performed the first approved gene therapy
procedure on four-year old Ashanthi DeSilva,
Born with a rare genetic disease called severe
combined immunodeficiency (SCID)

4. What did they do
• In Ashanthi's gene therapy procedure, Doctors
removed white blood cells from the child's
body, let the cells grow in the laboratory,
inserted the missing gene into the cells, and
then infused the genetically modified blood
cells back into the patient's bloodstream.

6. Types OfGene Therapy
• Gene therapy may be classified into two types:
1. Somatic gene therapy - the therapeutic genes are transferred into
the somatic cells, or body of a patient. Any modifications and
effects will be restricted to the individual patient only, and will not
be inherited by the patient's offspring or later generations
2. Germ line gene therapy - sperm or eggs, are modified by the
introduction of functional genes, which are integrated into their
genomes. This would allow the therapy to be heritable and passed
on to later generations

7. EX VIVO GENE THERAPY
• Grow the cells in culture
Transplant the modified cells to the patient.
Select genetically corrected cells and grow.
Introduce the therapeutic genes .
Grow the cells in culture
Isolate cells with genetic defect from a patient

8. IN VIVO GENE THERAPY
• Direct delivery of therapeutic gene into target
cell into patients body.
• Carried out by viral or non viral vector
systems.
• It can be the only possible option in
patients where individual cells
cannot be cultured in vitro in
sufficient numbers (e.g. brain cells).

11. Challenge of Gene therapy
• Gene delivery
• Entery of the therapeutic gene in the interior
of a cell

12. VECTORS IN GENE THERAPY
• To transfer the desired
gene into a target cell,
a carrier is required.
Such vehicles of gene
delivery are known
as vectors.
• 2 main classes
– Viral vectors
– Non viral vectors

13. • An ideal vector needs to meet three criteria:
• (1) it should protect the transgene against
degradation by nucleases in the extracellular
matrix
• (2) it should bring the transgene across the
plasma membrane into the nucleus of target
cells
• should have no detrimental effects

15. NONVIRAL VECTORS
• Physical approaches=use a physical force that
permeates the cell membrane to facilitate
intracellular gene transfer
• Needle injection
• Electroporation
• Gene gun
• Ultrasound
• Hydrodynamic delivery

16. METHODS OF GENE DELIVERY
Gene Gun
• Employs a high-pressure delivery system to shoot
tissue with gold or tungsten particles that are coated
with DNA
Microinjection
• Process of using a glass micropipette to insert
microscopic substances into a single living cell.
• Normally performed under a specialized optical
microscope setup called a micromanipulator.

17. Chemical approaches
• Use synthetic or naturally occurring
compounds as carriers to deliver the genetic
material into cells
• The liposomal delivery system
• Lipid polymer hybrid systems

18. CHEMICAL METHODS
• USING DETERGENT MIXTURES
– Certain charged chemical compounds like Calcium
phosphates are mixed with functional DNA of desired
function.
– The mixture is introduced near the vicinity of recipient cells.
– The chemicals disturbs the cell membrane, widens the pore
size and allows DNA to pass through the cell.
• LIPOFECTION
– It is a technique used to inject genetic materials into a cell by
means of liposomes.

19. • LIPOSOMES:
• These are lipid bilayers surrounding an aqueous vesicle
• Can be used to introduce foreign DNA into a target cell
• Advantages:
• Safer when compared to Viral vectors.
• Can carry large DNA molecules.
• Disadvantages:
• Inefficient transfer.
• Transient expression.

20. VIRAL VECTORS
• (1) Replication-defective vectors
• (2) Replication-competent vectors

21. Replication-defective vectors
• The vector is derived from a virus from which
all or most of the viral genes have been
removed to minimize virus-mediated toxicity
• Retro virus
• Herpes simplex virus (HSV)
• AV
• Reovirus
• Newcastle disease virus (NDV)

22. VIRAL VECTORS
• 1) RETROVIRUS VECTOR SYSTEM
• The recombinant retroviruses have the ability to integrate
into the host genome in a stable fashion.
• Can carry a DNA of
size – less than 3.4kb
• Replication defective
virus particles
• Disadvantages:
• Uncontrolled integration; May be oncogenic.
• Cannot infect non-dividing cells

23. • ADENO VIRUSES:
• Second most commonly used delivery system in gene therapy.
• Adenoviruses can be produced at high titres in cultures.
• Advantages:
• Can infect non-dividing cells,thus suitable for gene therapy of Cystic
fibrosis, DMD.
• Non-integration to chromosome. Avoids the risks of uncontrolled
integration.
• Efficient gene transfer.
• Disadvantages:
• Transient expression of gene due to episomal integration.
• Provokes immune response.

24. 2) ADENO VIRUS VECTOR SYSTEM
• Adeno virus with a
DNA genome
– good vectors.
• Target- non dividing
human cell.
• Eg. Common cold
adenovirus.

25. Replication-competent vector
• Selected viral genes are deleted or mutated so
that viral targeting or replication, or both can
occur selectively in tumor versus endogenous
neural cells.

29. Potential Areas for Gene Therapy in
the Brain
• Alzheimer’s disease
• Parkinson’s disease
• Brain tumours
• Ischemic brain diseases
• Epilepsy
• Amyotrophic lateral sclerosis

30. GENES DELIVERED INTO BRAIN
TUMORS
• (1) Pro drug activating (suicide gene therapy,
chemotherapy-sensitizing gene therapy, or
gene-directed enzyme prodrug therapy)
• (2) Transfer of tumor suppressor genes and
Cell cycle modulators
• (3) Genetic immune modulation
• (4) Anti angiogenic gene therapy

31. suicide gene therapy
• Involves transducing tumor cells with a gene
encoding an enzyme that can metabolize a
nontoxic prodrug to its toxic form
• Most commonly used technique in clinical
trials for brain tumors

32. HSV-1 Thymidine Kinase/Ganciclovir
• HSVtk is an enzyme that metabolizes nontoxic
nucleoside analogues,such as ganciclovir,
acyclovir, or valacyclovir into a cytotoxic
molecule= metabolite is incorporated into
DNA= cell death
• DNA polymerases inhibition

33. Bystander effect.
• The cytotoxic effect of transduced cells on
adjacent nontransduced cells is termed the
bystander effect.

34. • Transfer of toxic phosphorylated forms of
ganciclovir to nontransduced cells presumably
via gap junctions
• Targeting of mitotically active endothelial cells
in tumor vessels by the RV vector
• An immune-associated response against a
nonhuman protein,such as HSVtk, leading to
diffuse cell death that affects neighboring
nontransduced cells

35. A Phase III Clinical Evaluation of Herpes Simplex Virus Type 1 Thymidine
Kinase and Ganciclovir Gene Therapy as an Adjuvant to Surgical Resection and
Radiation in Adults with Previously Untreated Glioblastoma
MultiformeAuthor information
N. G. Rainov
Department of Neurosurgery, Martin-Luther Universität, Halle-Wittenberg, D-
06097 Halle, Germany
• a phase III, multicenter, randomized, open-label, parallel-group, controlled trial of
the technique in the treatment of 248 patients with newly diagnosed, previously
untreated glioblastoma multiforme (GBM). Patients received, in equal numbers,
either standard therapy (surgical resection and radiotherapy) or standard therapy
plus adjuvant gene therapy during surgery. Progression-free median survival in the
gene therapy group was 180 days compared with 183 days in control subjects.
Median survival was 365 versus 354 days, and 12-month survival rates were 50
versus 55% in the gene therapy and control groups, respectively. These differences
were not significant. Therefore, the adjuvant treatment improved neither time to
tumor progression nor overall survival time, although the feasibility and good
biosafety profile of this gene therapy strategy were further supported.

36. Selective Enhancement by an Antiviral Agent of the Radiation-induced Cell Killing of
Human Glioma Cells Transduced with HSV-tk Gene1
Jae Ho Kim2, Sang Hie Kim, Stephen L. Brown, and Svend O. Freytag
Henry Ford Hospital, Department of Radiation Oncology, Detroit, Michigan 48202-
4689
• The activation of antiviral drugs as a consequence of thymidine kinase expression
has been shown in recent years to have potential as a treatment for malignant
tumors. It was hypothesized that the property of the drugs that make them
effective against viruses and proliferating cells, namely their ability to interfere
with the integrity of the DNA, may be exploited to sensitize cells to radiation
damage. The antiviral drug, BVdUrd, structurally a pyrimidine analogue, was found
to enhance selectively the radiation cytotoxicity of human tumor cells transduced
with the HSV-tk thymidine kinase gene. Human glioma cells from the U-251
lineage transduced with HSV-tk and exposed to 40 µg/ml of BVdUrd for 24 h prior
to irradiation were more sensitive to radiation compared with control cells under
the same conditions; the sensitization enhancement ratio was 1.9. The results
suggest that the addition of radiation will improve the effectiveness of HSV-tk gene
therapy for the treatment of brain tumors.

37. Cytosine Deaminase/5-Fluorocytosine
• Cytosine Deaminase= 5-Fluorocytosin= 5-
fluorouracil
• The toxic effects of 5-FU are mediated by its
intracellular metabolites, which cause DNA
strand breakage leading to cell death

38. Cytochrome P-450
2B1/Cyclophosphamide
• The rat cytochrome P-450 2B1 (CYP2B1)
activates CPA with high efficiency
• HSV-1 vector (rRp450) that can kill tumor cells
through two modes:
• (1) using viral oncolysis
• 2) rendering the infected cell sensitive to CPA

39. Tumor Suppressor Genes
and Cell Cycle Modulators
• Transfer of functional gene controling cell
cycle in order to restore normal cycle
functioning
• The p53 pathway
• retinoblastoma protein (RB)/cyclin-dependent
kinase (CDK)/cyclin-dependent kinase
inhibitor (CDKN) circuit pathway

40. Trial of Adenovirus-Mediated p53 Gene Therapy for Recurrent Glioma:
Biological and Clinical Results Frederick F. Lang, Janet M. Bruner, Gregory N. Fuller,
Departments of Neurosurgery, Pathology and Neuro-Oncology, The University of Texas
M.D. Anderson Cancer Center, Houston, TX; Department of Neurosurgery, University
of California, San Francisco
• fifteen patients enrolled , patients underwent a two-stage approach. In stage 1,
Ad-p53 was stereotactically injected intratumorally via an implanted catheter. In
stage 2, the tumor-catheter was resected en bloc, and the postresection cavity was
treated with Ad-p53 Intratumoral injection of Ad-p53. However, at the dose and
schedule evaluated, transduced cells were only found within a short distance of
the injection site. Although toxicity was minimal, widespread distribution of this
agent remains a significant goal

41. Genetic Immune Modulation
• Genetic immune modulation enhances the immune
response against tumors by expressing cytokines and
lymphokines
• cytokines used frequently to achieve genetic immune
modulation of tumors are=
• interleukin-2
• interleukin-4
• interleukin-12
• interferon-β
• interferon-γ
• granulocyte-macrophage colony-stimulating factor

42. • Tumor cells are infected ex vivo with cytokine
genes
• RV producer cells carrying immune
modulating genes into the tumor so that
infection occurs in situ

43. Antiangiogenic Gene Therapy
• It has been shown that in vivo transfer of a
recombinant AV vector carrying the gene for
vascular endothelial growth factor in an
antisense orientation into gliomas inhibits
tumor growth

45. Gene Therapy for Parkinson’s Disease
• Currently, there are two ongoing gene therapy
clinical trials regarding Parkinson’s disease.
• One uses the approach of subthalamic GAD
(glutamate decarboxylase )gene transfer
• The other uses the approach of intrastriatal
gene transfer of ADDC (aromatic amino acid
decarboxylase)

46. Gene Therapy for Alzheimer’s Disease
• Most prominent systems affected in the
course of AD are the cholinergic neurons of
the nucleus basalis magnocellularis
• Two trials are ongoing based on NERVE
GROWTH FACTOR as a neuroprotective
molecule

47. First Real-Time MRI-Guided Gene Therapy for
Brain Cancer
Neurosurgeons at the University of California,
San Diego School of Medicine and UC San
Diego Moores Cancer Center are among the
first in the world to utilize real-time magnetic
resonance imaging (MRI) guidance for
delivery of gene therapy as a potential
treatment for brain tumors

48. IS GENE THERAPY TOTALLY SAFE ??
• Although gene therapy is a promising treatment
option for a number of diseases (including inherited
disorders, some types of cancer, and certain viral
infections), the technique remains risky and is still
under study to make sure that it will be safe and
effective.
• Gene therapy is currently only being tested for
the treatment of diseases that have no other
cures

49. Problems with Gene Therapy
• Short Lived
– Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells
prevent gene therapy from long time
– Would have to have multiple rounds of therapy
• Immune Response
– new things introduced leads to immune response
– increased response when a repeat offender enters
– the gene might be over-expressed (toxicity)
• Viral Vectors
– patient could have toxic, immune, inflammatory response
– also may cause disease once inside