basics of cancer gene therapy

1. Dr / Ahmad Abdel hady
Assistant lecturer of clinical oncology

2. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

3. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

4. Gene therapy involves the introduction of
exogenous nucleic acids (usually DNA) into cells with a
view to alter the phenotype of a diseased cell via altered
patterns of gene expression.
WHAT
?????

5. Exogenous nucleic acid
Consists of
 The transcription unit of a therapeutic gene.
 Regulatory sequences controlling transcription
initiation and termination.

6. Function
 Replace missing/damaged cellular chromosomal DNA
sequences.
 Introduce novel gene products.
SO
 restore the normal phenotype .
 OR kill the diseased cell.

7. Aim of gene therapy
Treatment of
Monogenic inherited
disorders
 Hemophilia.
 severe combined immunodeficiency.
 cystic fibrosis.
Acquired diseases
 Vascular disease (including coronary artery and cerebro-
vascular disease).
 Neurological diseases such as Parkinson’s disease.
 Infectious diseases, particularly acquired immune

8. Cancers are attractive targets for gene therapy.
Cancer gene therapy has the dual aims
 enhancing recent improvements in cancer survival
rates, achieved by existing cancer treatments.
 with maximum efficacy and minimum toxicity
(optimum therapeutic index).

9. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

10. A.Tumor cells themselves
So they can
 Replace defective tumor suppressor genes.
 Down-regulate over-expressed cell surface receptors
(or inhibit their kinase activity).
 Block aberrant intracellular signaling pathways.
 Restore the cells’ capacity to undergo senescence.
 Stimulate apoptosis .
 Inhibit tumor angiogenesis.

11. B. normal cells,
 For example, stem cells, which are sensitive to
the anti-proliferative effects of conventional cytotoxic
drugs.
 can be selectively protected by incorporating drug
resistance genes, permitting the use of
chemotherapy in a more dose-intense fashion, without
affecting sensitive normal cells.

12. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

13. ‘gene replacement’:
aim to correct the molecular defects which result in
malignant transformation
‘suicide genes’:
where phenotypic reversal is difficult or impossible,
the tumor cells may be selectively killed by the
expression of so-called ‘suicide genes’

14. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

15. Route of delivery
 Trans-gene can be delivered to tumor insitu by direct
intra-tumoral injection.
 Or ……….trans-gene can be incorporated into cells ex-
vivo with cells being extracted from tumor-bearing
patient , the trans-gene introduced into cells extra-
corporeally, and the genetically modified cells
returned to the host .

16.  Ex-vivo – adoptive – transfer:
Useful approach for
1. Hematological malignancies (peripheral blood stem
cells).
2. immunotherapy .
The idea of natural tropism to the site of the tumor
Either bone marrow or cells of immune system.

17.  The previous way is not applicable for solid tumors,
an alternative way of administration for superficial
lesions and where the tumor is
localized & not metastasized
 S.C. lesions
Tumors visualized by endoscope , where intra-tumoral
inj. Of naked DNA can be performed under direct
vision (Gene Gun)

18. What is the value of localized gene therapy ?
Especially when the existing local treatments like
surgery or radiotherapy can achieve good cure rates.
It has no added value
Gene therapy is most likely has a great value in
treatment of advanced disease with multiple
metastases, where the best route of delivery is usually
intravenous and occasionally into arteries.

19. Limitations of systemic gene therapy
Main obstacles to successful implementation of gene
therapy for disseminated malignancy are :
1. Circulating antibodies and immune effector cells.
2. Non-specific adsorption in liver.
3. Extravasation and interaction with ECM
(extracellular matrix).

20. How to overcome these problems?
 Deliver trans-gene inside autologus cells which have
the intrinsic ability to home to sites of tumor growth,
These cells like
Lymphocytes.
Macrophages .
Endothelial cells.
Mesenchymal stem cells.
Hiding the trans-gene within these cells

21. Then to dissemination of the trans-gene throughout the
tumor once insitu using
 Antigen recognition.
 Pharmacological stimulus.
To release the trans-gene from cellular carrier

22. GENE DELIVARY SYSTEMS
BIOLOGICAL CHEMICAL PHYSICAL
GENETICALLY
MODIFIED VIRAL
VECTORS
GENERATION OF
LIPOSOMES FROM
CATIONIC LIPIDS OR
POLYMERS
DIRECT
INJECTION
With assistance of
hydrodynamic tech.,
U.S. treatment , or
electroporation

23. Viral vectors
What are the criteria of ideal viral vector?
1. Non-pathogenic.
2. Non-immunogenic.
3. Capable of production of high titre to permit
saturation of target cells.
4. Packing capacity sufficient to include coding&
regulatory sequences of trans-gene.
5. Ability to gain entry to non-dividing cells as well as
proliferating cells.

24. Various families have been assayed for this use ……
Non of them have all these criteria….
 Retrovirus vectors.
 Lentivirus (HIV-1 and 2) vectors.
 Adenovirus vectors.
 Adeno-associated virus vectors.
 Herpes virus vectors.
 Hybrid virus vectors.

25. ONCOLYTIC VIRUS
 A class of viral vectors which replicate preferentially in
tumour cells.
 It’s also called conditionally replicating virus.
 Their main advantages
1. Selectively target tumour cells.
2. Improve the spread of trans-gene products from cell
to cell so increase efficiency of gene therapy.
3. In addition they exploit the defects in intracellular
pathways which are commonly associated with
tumour phenotype.

26.  Newcastle disease virus& vesicular stomatitis virus are
tumour selective by exploiting defects in interferon
response pathways.
 Reovirus exploit the activated Ras pathway for
replication.
Some viruses are not oncolytic but can be genetically
engineered to exploit features associated with tumour
cells.

28. Non-viral gene delivery
Advantages :
1. Absence of viral coat improve safety.
2. Reduced immunogenicity.
3. Avoid regeneration of potential pathogenic wild type
of virus or insertional mutagenesis or oncogenesis.

29. Physical method
 Use of naked DNA directly has the following
advantages,
1. Simple.
2. Inexpensive .
3. Non-immunogenic.
4. There is no limitation as regard to packing ….i.e
there is no size limits on trans-gene or its regulatory
sequence.

30.  Simplest method is injection by syringe or needle (bio-
ballistic delivery system or gene gun)
DNA plasmid coated with GOLD particles and pushed
via inert gas as an alternative (needle-free mechanism)
for local injection.

31.  With assistance to increase permeability of cell
membrane to naked DNA by local application of
1. U.S. waves
2. Electroporation(low strength electromagnetic waves).
 Rapid injection (hydrodynamic) of large volume of DNA
solution mechanical stretch of endothelium.
 Transient localized occlusion of blood vessels such as
hepatic portal vein immediately after bolus
administration.

32. Chemical method
 Enhancing uptake of DNA plasmid is to complex DNA with
poly-cations forming liposomes with cationic lipids ……
 1st generation DOPE or DOTAP
 2nd generation PLL or PEI
The latter acts as a proton sponge , influx of protons into
endosomes bring water with it rupture of endosomes
& release of DNA in cytoplasm.
 Once in the nucleus, for long term expression of trans-gene
chromosomal integration facilitated by
bacteriophage site-specific integrase enzyme (SLEEPING
BEAUTY)

33.  Naked DNA & DNA/liposome complex are less
immunogenic than many viral vectors.
 But still can generate limited immune response.
 This can be reduced by removal of CpG motifs from
DNA plasmid

34. Artificial Virus Like
Particles
AVLP

35.  For better efficiency uptake and subsequent
expression
 Combine best features of both viral and non-viral
vectors.
 Plasmid DNA packed within synthetic polycation
coating.
 Improved cell entry achieved by conjugation with
eukaryotic or viral peptides role in process of
receptor binding & internalization.

36. Specificity in targeting of AVLP can be achieved by
incorporating
Specific
ligands
Non- specific
ligands
Monocolonal
antibody against
transferrin receptors
Peptides containing
integrin binding RGD
peptide sequence

37.  Transcriptional targeting.
Tissue specific promoters
Tumor selective promoters
Inducible promoters
 Cell surface targeting.
 Oncolytic virus.

38. Transcriptional targeting
Most gene therapy strategies involve a trans-gene
mRNA intermediate
effector protein
DNA-directed RNA polymerase

39. Promoters & enhancers
 Transcription regulatory sequences.
 Determine
1- site of initiation of transcription.
2- quantity of transcripts produced from the gene
 The initiation of transcription must be tightly
regulated and the product of trans-gene must be
expressed only when (temporal control) & where
(spatial control) intended.

40. 1.TISSUE SPECIFIC PROMOTERS
 For percise patial control.
 Especially after systemic administration of gene
therapy construct …..to make sure that subsequent
transcription and gene expression will occur only in
specific tissue .
 Promoter region sequence recognized by transcription
factors found only in particlar tissue without which
transcription initiation complex cannot be performed.

41. Gene which is expressed
only in a particular tissue
pSuicide gene
Suicide gene
p

42. On this basis
1. Tyroinase gene promoter restrict expresssion to
melanocytes in treatment of melanoma
2. Alfa-1 antitrypsin & albumin gene promoter to target
liver cells in hepatoma.
3. Glial Fibrillary Acidic Protein promoter (GFAP) to
target astrocytes in CNS.
4. Immunoglobulin promoters to B lymphocytes in B
cell lymphoma

43. 2. tumour selective
promoters
 The previous type of promoter carry the risk of
affection to normal tissue.
 Tumour selective promoter avoid this problem as they
target tumour cells only
 E.g. Onco-fetal protein and alfa fetoprotein promoter
which expressed only in HCC & Testicular teratoma.
 CEA promoter which target colorectal cancer cells and
some other cancers.
 PSA gene promoter for prostate cancer cells .
 Thyroglobulin promoter for thyroid cancer.

44.  It would be desirable to have a single gene therapy
construct with broad tumour tropism.
Human telomerase
 Over-expressed in 80-90% of human tumours
 Its role is to permit escape from overgrowth and
permits cellular death.
 When we include the RNA or protein component of
human telomerase provide tumour selective
gene therapy for wide range of tumour types.

45. 3. Inducible
promoters
 Used for temporal control of gene therapy as the previous 2
type are used mainly for spatial control.
 The mean of temporal control of gene therapy is the ability
of multiple dosing of gene therapy after single
administration of the gene construct.
How is that?
By addition of a small molecule which enable us to switch the
transcription ON & OFF (GENE SWITCH SYSTEM).
E.g.
1. Heavy metal ions (zinc & cadmium) metallothionein
promoters.
2. Tetracyclin or its analogue doxycycline.

46. 4. Other
promoters
 Some promoters are active only under certain
physiological conditions depending on tumour micro-
environment.
e.g. Hypoxia inducible promoters
Transcription of genes encoding for products such as
1. Erythropoietin.
2. VEGF.
3. Carbonic anhydrase IX.
Useful in combination with radiotherapy (Genetic
Radiotherapy)

47. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

48. a) Gene replacement strategies.
b) Suicide gene therapy.
c) RNA-directed strategies.

49. Gene replacement strategies
 Loss of both copies of recessive suppressor genes
observed in many human tumours.
 So the principle is to re-introduce a single functioning
copy to reverse or abolish the tumour cell phenotype.
 The most attractive target for gene replacement is P53
the guardian of the genome.

50. Suicide gene therapy
 In its simplest form ..........it encodes for protein such
as A-subunit of Diphtheria toxins......direct cellular
cytotoxicity.
 But commonly suicide gene product is not toxic in
itself
Drug with cytotoxic
activity
Non toxic pro-drug Enzyme

51.  2 types of suicide genes therapy protocols:
1. Gene directed enzyme pro-drug therapy (GDEPT)
2. Virus directed enzyme pro-drug therapy (VDEPT).
The first pro-drug activating enzyme used is Bacterial
cytosine de-aminase CD.
De-aminate 5 F.C. ...............TO ...... 5 F.U.

52.  The most widely used is herpes-simplex virus
thymidine kinase (HSV-tk) gene.
 It activate the pro-drug Ganciclovir by facilitating the
triphosphorylation steps.
 Triphosphate is a DNA replication chain
terminator in cell cycle S-phase.
 The HSV-tk only facilitate the 1st step phosphorylation
producing monophosphate (which cannot be done in
mammalian cells), while the di & tri phosphorylation
are done by endogenous kinases.

53. RNA-directed strategies
 In stead of targeting DNA coding for protein or the
protein itself , these strategies aim to target mRNA.
 E.g. Targeting mRNA of EGFR in EGFR-over expressed
tumours.
There are 3 types
1. Anti-sense technology.
2. Ribozymes.
3. RNA interference.

54. 1- Anti-sense technology:
 Single-stranded oligo-deoxynucleotides (ODNs),
complementary to mRNAs can reduce the level of
expression of the encoded protein in one of two ways:
Either – sequence specific base pairing between
antisense ssODN and targeted mRNA inhibiting its
translation.
Or – ssODN form RNA: DNA hetero-duplex which
recognized by cellular enzyme RNase H leading to
mRNA cleavage.

55. 2- Ribozyme:
 Derivative of naturally occurring molecules found in a
variety of organisms ciliated protozoan Tetrahymena
thermophila.
 Like ODNs bind to target mRNA
 Still under research on AIDS and healthy volunteers
not for cancer patients.

56. 3- RNA- Interference or Gene Silencing:
 Achieved by 21-23 nucleotide base ssRNA effectors
which is called siRNA which is complementary to
mRNA targets.
 siRNA production is via an intracellular multi -
molecular complex known as RISC (RNA-induced
silencing complex).

58. Introduction
Components of a gene therapy
strategy
Cellular targets for gene therapy
Effector mechanism
Delivery strategy
Effector gene
Gene therapy in practice.

59. An individualized strategy for each clinical situation
should be based upon an understanding of the
biological behavior and natural history of the tumor.
Each strategy has multiple components—choice of
target, Effector mechanism for altering the tumor cell
phenotype, delivery strategy, tumor-selective targeting
of trans-gene expression, and the nature of the
Effector trans-gene itself.

60.  Humanised monoclonal antibodies have been
demonstrated in Phase III clinical trials to improve
survival in subsets of breast cancer (Slamon et al.,
2001) and colon cancer.
 while receptor tyrosine kinase inhibitors have been
shown to be effective in chronic myelogenous
leukaemia and gastrointestinal stromal tumours.
 A phase I trial of adenovector-mediated delivery of
interleukin-2 (AdIL-2) in high-risk localized prostate
cancer (Suzane Trudel et al.,2003)

61.  Phase II/III trials of p53-expressing adenoviral vectors,
given intravenously or intraperitoneally in combination
with systemic chemotherapy/ radiotherapy are now
under way in NSCLC, head-and-neck cancer and ovarian
cancer (Gene Therapy Clinical Trials Database, 2004).

62. Thank you
See you in cancer gene therapy
to individual systems soon