Novel approach to treat, cure or ultimately prevent a disease by changing the expression of a person’s genes

1. By: LARAIB
M.Phil. pharmacology

2. GENE THERAPY
Novel approach to
treat, cure or
ultimately prevent a
disease by
changing the
expression of a
person’s genes

3. Concept of gene therapy
 originally referred to proposed treatments of
genetic disorders
 involve replacing a defective gene with its
normal counter-part
 The first approved gene therapy experiment
occurred on September 14, 1990 in US, when
Ashanti DeSilva was treated for ADA-SCID

4. STEPS IN GENE THERAPY:
Identification of the defective gene
Cloning of normal healthy gene
Identification of target cell / tissue / organ
Insertion of the normal functional gene into the host
DNA.

5. Approaches involved in gene
therapy
 Replacing a mutated gene that causes disease
with a healthy copy of the gene
 Inactivating, or “knocking out,” a mutated gene
that is functioning improperly
 Introducing a new gene into the body to help
fight a disease

6. Researchers are studying gene therapy for a number
of diseases, such as
• Severe combined immuno-deficiencies (SCID)
• Hemophilia
• Parkinson's disease
• Cancer
• HIV

7. How It Works
A vector delivers
the therapeutic
gene into a
patient’s target
cell
The target
cells become
infected with
the viral
vector
The vector’s
genetic
material is
inserted into
the target cell
Functional proteins
are created from
the therapeutic
gene causing the
cell to return to a
normal state

9. APPROACHES INVOLVED IN GENE
THERAPY
Germ line gene therapy:
 where germ cells (sperm or egg) are modified by
the introduction of functional genes, which are
integrated into their genome.
 Therefore changes due to therapy would be
heritable and would be passed on to later
generation.
 Theoretically, this approach should be highly
effective in counteracting genetic disease and
hereditary disorders.

10. Somatic gene therapy:
 where therapeutic genes are transferred into
the somatic cells of a patient.
 Any modifications and effects will be restricted
to the individual patient only and will not be
inherited by the patients offspring or any later
generation.

11. Types of somatic gene therapy
2. In vivo
approach
1. Ex vivo
approach
somatic gene
therapy

12. EX VIVO GENE THERAPY

14. Example of ex vivo gene therapy
 1st gene therapy – to correct deficiency of enzyme,
Adenosine deaminase (ADA).
 Performed on a 4yr old girl Ashanthi DeSilva.
 Caused due to defect in gene coding for ADA. Deoxy
adenosine accumulate and destroys T lymphocytes.
 Disrupts immunity , suffer from infectious diseases and
die at young age

16. IN VIVO GENE THERAPY
 Direct delivery of therapeutic gene into target
cell into patients body
 it is only possible option
in patients where
individual cells
cannot be cultured
in vitro in sufficient
numbers (e.g. brain cells).

17. Carried
out by
viral or
non viral
vector
systems

18. Example of in vivo gene therapy
-Therapy for cystic
fibrosis
 A protein, cystic fibrosis transmembrane regulator
(CFTR) is absent due to a gene defect
 In the absence of CFTR chloride ions concentrate
within the cells and it draws water from
surrounding.
 This leads to the accumulation of sticky mucous in
respiratory tract and lungs.
 Treated by in vivo replacement of defective gene
by adenovirus vector .

20. 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

22. VIRAL VECTORS
 Viruses have evolved a way of encapsulating
and delivering their genes to human cells to
remove disease-causing genes and insert
therapeutic ones
 Virus bind to their hosts and introduce their
genetic material into the host cell

24. Retrovirus vector system
 The recombinant retroviruses have the ability to
integrate into the host genome in a stable fashion
 They infects a host cell, and introduce its RNA together
with some enzymes, namely reverse transcriptase and
integrase, into the cell
 DNA copy is produced from RNA and is free in the
nucleus of the host cell, and must be incorporated into
the genome of the host cell

26. Adenovirus vector system
 Contains linear double stranded DNA
 Does not integrate into the host genome
 The extra genes are not replicated when the cell is
about to undergo cell division so the descendants of
that cell will not have the extra gene
 treatment with the adenovirus will require
readministration in a growing cell population

27.  the absence of integration into the host cell's
genome should prevent the type of cancer seen
in the SCID trials
 This vector system has been promoted for
treating cancer and indeed the first gene
therapy product to be licensed to treat cancer

29. Adeno-associated virus vector
 AAV is a simple, non-pathogenic, single
stranded DNA virus dependent on the helper
virus (usually adenovirus) to replicate
 AAV enters host cell, becomes double
stranded and gets integrated into chromosome

30. Herpes Simplex Viruses
 Double stranded DNA
viruses that infect neurons
 This is mostly examined for gene transfer in
the nervous system

31. Advantages of viral vectors:
They're very good at targeting and entering cells.
Some target specific types of cells.
They can be modified so that they can't replicate
and destroy cells.

32. Drawbacks of viral vectors:
 They can carry a limited amount of genetic material.
Therefore, some genes may be too big to fit into some
viruses.
They can cause
immune
responses in
patients, leading
to two potential
problems:
Patients may get
sick.
The immune system
may block the virus
from delivering the
gene to the patient's
cells, or it may kill
the cells once the
gene has been
delivered.

33. Non viral vector system
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

34. Naked DNA
 It is simplest method of injecting naked DNA into
the human body
 In this method naked plasmid DNA was used for
injecting intramuscularly
 Relatively low level of expression and but
sufficient for use in DNA vaccination.

35. PHYSICAL METHODS TO ENHANCE
GENE DELIVERY
1. Gene gun:
 In this method DNA is coated
with gold particles and loaded
into a device
 Employs a high-pressure
delivery which is similar to
gun and it generates force
by which it can penetrate into
the cell

36. 2. Electroporation:
 Short pulses of high voltage carry DNA across the cell
membrane
 This cause temporary formation of pores and thus allow
DNA molecules to pass
Nucleus
DNA enters

37. 3. Sonoporation:
 ultrasonic frequencies are used to deliver DNA into cells.
 Which can disrupt the cell membrane and allow DNA to
move into cells.

38. 4. Magnetofection:
 DNA is made into magnetic particles, and a magnet is
placed underneath the tissue culture dish to bring DNA
complexes into contact with a cell monolayer.

39. 5. Hydrodynamic delivery
 Hydrodynamic delivery involves rapid injection of a
high volume of a solution into vasculature (such as
into the inferior vena cava, bile duct, or tail vein)
 The solution contains molecules that are to be
inserted into cells, such as DNA plasmids or siRNA,
 transfer of these molecules into cells is assisted by
the elevated hydrostatic pressure caused by the
high volume of injected solution

40. CHEMICAL METHODS TO ENHANCE GENE
DELIVERY
Oligonucleotides
 The use of synthetic oligonucleotides in gene therapy is
to deactivate the genes involved in the disease
process.
There are several methods by which this is achieved.
 One strategy uses antisense specific to the target gene
to disrupt the transcription of the faulty gene.
 Another uses small molecules of RNA called siRNA to
signal the cell to cleave specific unique sequences in the
mRNA transcript of the faulty gene, disrupting translation

41. Polymersomes
 Polymersomes are synthetic versions of liposomes
made of amphiphilic block copolymers.
 They can encapsulate either hydrophilic or
hydrophobic contents and can be used to deliver
DNA, proteins, or drugs to cells.
 Advantages of polymersomes over liposomes
include greater stability, mechanical strength, blood
circulation time, and storage capacity.

42. HYBRID METHODS
 Due to every method of gene transfer having
shortcomings, there have been some hybrid methods
developed that combine two or more techniques
Virosomes are one example; they combine liposomes
with an
inactivated HIV or influenza virus.
 This has been shown to have more efficient gene
transfer in respiratory epithelial cells than either viral or
liposomal methods alone.
 Other methods involve mixing other viral vectors with

43. VARIOUS STRATEGIES FOR GENE
THERAPY:
1. Gene augmentation therapy
2. Inhibition of gene expression
3. Gene therapy to achieve pharmacological
effects/
killing specific cells

44. GENE AUGMENTATION THERAPY:
 Foreign gene replaces missing or defective
gene.
 This is used to treat diseases caused by a
mutation that stops a gene from producing a
functioning product, such as a protein
 Eg. Replacement of defective p53 gene by a
normal one in liver cancer.

46. INHIBITION OF GENE
EXPRESSION:
 Suitable for the treatment of infectious diseases,
cancer and inherited disease caused by
inappropriate gene activity.
 eliminate the activity of a gene that encourages
the growth of disease-related cells.

48. Killing of specific cells
 The aim is to insert DNA into a diseased cell that
causes that cell to die.
 This can be achieved in one of two ways:
the inserted DNA contains a “suicide” gene that
produces
a highly toxic product which kills the diseased cell
 the inserted DNA causes expression of a protein
that marks the cells so that the diseased cells are
attacked by the body’s natural immune system.

50. APPLICATIONS OF GENE THERAPY
Gene Therapy for Genetic Disorders
Severe Combined Immune Deficiency (ADA-SCID)
Affected children are born without an effective immune
system and will succumb to infections
The disease is caused by a mutation in a
gene on chromosome 20. The gene
codes for the enzyme adenosine
deaminase (ADA).

53.  The therapeutic gene called ADA was
introduced into the bone marrow cells of such
patients in the laboratory, followed by
transplantation of the genetically corrected cells
back to the same patients.
 The immune system was reconstituted

54. Chronic Granulomatus Disorder
(CGD)
 CGD is a genetic disease in the immune system
 that leads to the patients' inability to fight off
bacterial and fungal infections that can be fatal.
 investigators in Germany treated two patients with
this disease some of the blood-making cells are
taken from the patient
 The normal gene is placed into the cells using
special viruses called retroviruses. The cells are
then able to produce the normal protein.

55. Hemophilia
 Patients are not able to induce blood clots and suffer
from external and internal bleeding that can be life
threatening.
 In a clinical trial conducted in the United States , the
therapeutic gene was introduced into the liver of
patients, who then acquired the ability to have normal
blood clotting time.
 The therapeutic effect was transient because the
genetically corrected liver cells were recognized as
foreign and rejected by the healthy immune system in
the patients and curative outcome by gene therapy

56. Diabetes
 Type I diabetes is an autoimmune disease
wherein the immune system erroneously targets
and destroys the insulin secreting beta cells of the
pancreas
 as a consequence requires patients with this
disease to treat themselves with daily injections of
insulin to control their blood sugar levels.
 To treat this disease, gene therapy investigators
are currently studying approaches to efficiently
transfer the insulin gene into other cells such as
the liver, stomach, or intestines.

57. GENE THERAPY TRIAL FOR INHERITED
BLINDNESS
 Choroideremia is a rare inherited cause of blindness that
affects around 1 in 50,000 people.
 There is currently no cure.
 It is caused by defects in the CHM gene on the X
chromosome.
 Without the protein produced by the CHM gene, pigment
cells in the retina of the eye slowly stop working, then
die off.

59.  The results showing improvement in vision in the
first six patients confirm that the virus can deliver its
DNA payload without causing significant damage to
the retina.
 This has huge implications for anyone with a
genetic retinal disease such as age-related macular
degeneration or retinitis pigmentosa, because it has
for the first time shown that gene therapy can be
applied safely before the onset of vision loss.’

60. Gene Therapy for Acquired
Diseases
Cancer
 Multiple gene therapy strategies have been developed to
treat a wide variety of cancers, including suicide gene
therapy, oncolytic and therapeutic gene vaccines.
 Two-thirds of all gene therapy trials are for cancer and many
of these are entering the advanced stage, including a Phase
III trial
 Additionally, numerous Phase I and Phase II clinical trials for
cancers in the brain, skin, liver, colon, breast and kidney
among others, are being conducted in academic medical
centers and biotechnology companies, using novel
technologies and therapeutics developed on-site.

61. Oncolytic virus HF10 project
 Live viruses such as HSV infect human cells,
replicate, and destroy the infected cells.
 Using this property of viruses, the new approach to
cancer therapy termed "oncolytic virotherapy" has
been developed and investigated.
 HF10 is one of the promising new strains for oncolytic
virotherapy.
 HF10 shows a strong killing effect against tumor cells
because of its high replication competence in these
cells.

62.  administration of HF10 also induces a strong immune
response which is expected to further enhance the anti-
tumor activity

63. TCR gene therapy project
 T-cell receptor genes which recognize cancer antigens
are transduced into lymphocytes of a cancer patient for
treatment.
 The gene-modified lymphocytes are cultured in a large
scale, and infused back into the patient.
 The lymphocytes express the T-cell receptors on the
surface, and these receptors recognize the peptides
derived from the cancer antigens. So the gene-modified
lymphocytes can specifically attack tumor cells
expressing the cancer antigens and kill them finally.

65. Neurodegenerative Diseases
PARKINSON’S disease
 uses a modified virus to deliver three genes into the
striatum, a part of the brain that controls movement.
 The genes are intended to boost the production of
dopamine, a chemical that becomes deficient in
patients with Parkinson’s.
 The gene copies enable the cells to pump out more
GABA

67. Huntington disease
 Gene silencing involves using a specially
designed drug to intercept a message molecule,
called RNA, that’s produced from the HD gene
and tells cells to make the harmful Huntington
protein.
 The effect of the drug is that cells make less of
the protein

69. Other acquired diseases
 The same gene therapeutic techniques have
been applied to treat other acquired disorders
such as viral infections (e.g. influenza, HIV,
hepatitis), heart disease and diabetes, among
others.
 Some of these have entered, or will soon be
entering, into early phase clinical trials.

70. Problems assosiated with Gene
Therapy
Due to rapid dividing of cells it is short lived
Immune response to the transferred gene
stimulates a potential risk to gene therapy.
Viruses used as vectors for gene transfer may
cause toxicity, immune responses, and
inflammatory reactions in the host.
Disorders caused by defects in multiple genes
cannot be treated effectively using gene therapy.

71. Ethical issues surrounding gene
therapy
 Current gene therapy research has focused on treating
individuals by targeting the therapy to body cells such as
bone marrow or blood cells. This type of gene therapy
cannot be passed on to a person’s children.
 Gene therapy could be targeted to egg and sperm cells
(germ cells), however, which would allow the inserted
gene to be passed on to future generations. This
approach is known as germline gene therapy.
 The idea of germline gene therapy is controversial.

72.  While it could spare future generations in a family
from having a particular genetic disorder, it might
affect the development of a fetus in unexpected
ways or have long-term side effects that are not yet
known.
 Because people who would be affected by germline
gene therapy are not yet born, they can’t choose
whether to have the treatment. Because of these
ethical concerns, the U.S. Government does not
allow federal funds to be used for research on
germline gene therapy in people.

73. Conclusion
 Theoretically, gene therapy is the permanent
solution for genetic diseases.
 But it has several complexities. At its current
stage, it is not accessible to most people due to
its huge cost.
 A breakthrough may come anytime and a day
may come when almost every disease will have
a gene therapy

75. Initial Clinical Results With Direct Myocardial Injection
of phVEGF165 as Sole Therapy for Myocardial Ischemia
Background:
 a phase 1 clinical study was conducted to determine the safety
and bioactivity of direct myocardial gene
transfer of vascular endothelial growth factor (VEGF)
as sole therapy for patients with symptomatic
myocardial ischemia

76. Methods
 Five patients with chronic, severe angina underwent
direct myocardial gene transfer
 All patients received eukaryotic expression vector
encoding
the 165-amino acid isoform of the human VEGF gene
Plasmid DNA encoding vascular endothelial growth factor
(VEGF) (125 ug) was administered by direct myocardial
injection in 4 aliquots of 2.0 mL each

77.  Patients underwent diagnostic angiography ,1 month
before and 60 days after gene transfer.

78. Results
 All patients underwent successful myocardial gene
transfer
 Injections caused no changes in heart rate, systolic
blood pressure or diastolic BP
 Serial ECGs showed no evidence of myocardial
infarction in any patient
 All 5 patients experienced a decrease in anginal
frequency and severity

79.  mean number of normally perfused segments per patient
increased from 6.061.1 before gene transfer to 8.060.7 (P,0.05) at
day 60 after gene transfer

80. Discussion
 The finding that VEGF could be used to achieve
angiogenesis that was therapeutic was first
demonstrated by Takeshita et. al
 this is a particularly appealing strategy because the
VEGF gene encodes a signal sequence which permits
the protein to be naturally secreted from intact cells

81.  The present study provides the first evidence for a
favorable clinical effect of direct myocardial injection of
naked plasmid DNA encoding for VEGF
 Each patient experienced a reduction in anginal
symptoms and nitrate use, and there is objective
evidence for reduced ischemia by perfusion imaging