Gene Therapy

Presented by:
1.Mahbubul
Chowdhury
2. Asma Hossain
3.Imrana Tasnim
4.Junaid shamim
 Introduction
 Gene therapy is the insertion of genes into
an individual cells and tissues to treat a
disease in which a defective mutant allele is
replaced with a functional one.

 DNA is used as a therapeutic agent.

 Genetic diseases, hematological disorders,

acquired immunodeficiency syndromes,
cancers are mainly treated.
 HISTORY AND DEVELOPMENT
OF GENE THERAPY
 1960: The concepts of Gene Therapy was introduced
 1970: Friedmann and Roblin author of a paper in Science
titled "Gene therapy for human genetic disease?” cite the
first attempt to perform gene therapy
 1990:
 The first approved gene therapy case at the National
Institute of Health, U.K. It was performed on a four year
old girl named Ashanti DaSilva. It was a treatment for a
genetic defect that left her with an immune system
deficiency
 New gene therapy approach repairs errors in
messenger RNA derived from defective genes. This
technique has the potential to treat the blood disorder
Thalassaemia, Cystic fibrosis, and some cancers
 Sickle cell disease is successfully treated in mice
 1992: Doctor Claudio Bordignon working at the Vita-Salute San
Raffaele University, Milan, Italy performed the first procedure of
gene therapy using hematopoietic stem cells as vectors to deliver
genes intended to correct hereditary diseases
 1999: Death of Jesse Gelsinger in a gene-therapy experiment
resulted in a significant setback to gene therapy research in the
United States
 2006: Scientists at the National Institutes of Health (Bethesda,
Maryland) have successfully treated metastatic melanoma in two
patients. This study constitutes one of the first demonstrations that
gene therapy can be effective in treating cancer.
 2007- 2011: Research is still ongoing and the number of diseases
that has been treated successfully by gene therapy increases.
 Retinal disease
 Colour blindness
 Adrenoleukodystrophy
 2011: Medical community accepted that it can cure HIV as in
2008, Gero Hutter has cured a man from HIV using gene therapy
 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
 STEPS IN GENE THERAPY:
The basic steps of gene therapy include:
The faulty gene that causes a specific condition must be
identified.
The location of the affected cells in the body's tissues or
organs must be pinpointed.
A working version of the gene must be available.
The working version of the gene has to be delivered to the
cell.
 Types of gene therapy
Germ line gene therapy Somatic cell gene therapy
 Germ line gene therapy

 Introduction of the foreign gene into germ

cells like sperm / ovum / fertilized egg.

 Results in expression of modified features in

both somatic as well as germ cells of the
offspring.

 Considered unethical, and is not advocated

in humans.
 Somatic cell gene therapy

 Insertion of therapeutic gene into somatic

cells like
fibroblasts,myoblasts,epithelial cells,
nervous cells,glial cells etc.

 This can correct the genetic defect in the

patient

 However,in somatic cell therapy, Transgene

cannot be passed on to the siblings etc.
Two ways to deliver genes:
1. Ex vivo approach
2. In vivo approach

EX VIVO
IN VIVO
 Ex vivo approach:
 Target cells are removed
from the body and grown
in vitro.
 The gene is then
introduced into the
cultured cells.
 These cells are then re-
introduced into the same
individual.
 Examples: Fibroblast
cells, Hematopoietic cells.
 EXAMPLE OF EX VIVO GENE THERAPY
 1st gene therapy – to correct deficiency of enzyme,
Adenosine deaminase (ADA).

 Performed on a 4yrs old girl Ashanthi DeSilva.

 Was suffering from SCID- Severe Combined

Immunodeficiency.

 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.
 In vivo approach:
Direct Gene Transfer

 Cloned therapeutic
gene is introduced
directly into the
affected tissue, without
removing cells from the
body.
 Specially designed
vehicles are needed.
 Examples are: Lungs,
Brain.
 EXAMPLE OF IN VIVO GENE THERAPY

 In patients with cystic fibrosis, a protein called 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 .
 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.

Two main classes-

Viral vectors
Non viral vectors
 VIRAL VECTORS
1) RETROVIRUS VECTOR:

 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
 Retroviruses are used ONLY in EX VIVO THERAPY.
Advantages:
• Chromosomal integration & stable modification of target
cells.

Disadvantages:
• Uncontrolled integration; May be oncogenic.
• Cannot infect non-dividing cells.
 VIRAL VECTORS
2) ADENO VIRUS VECTOR:

 Second most commonly used

delivery system in gene
therapy.

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.
 VIRAL VECTORS
3) ADENO ASSOCIATED VIRUS VECTOR:
 It is a human virus that can integrate into
chromosome19.

 It is a single stranded, non pathogenic small DNA virus.

 AAV enters host cell, becomes double stranded and

gets integrated into chromosome.

4) HERPEX SIMPLEX VIRUS VECTOR:

 Viruses which have natural tendency to infect a
particular type of cell.
 They infect and persist in nervous cells.
 NON VIRAL VECTORS
1) PURE DNA CONSTRUCT:

– Direct introduction of pure DNA construct into target tissue .

– Efficiency of DNA uptake by cells and expression rather low.

– Consequently, large quantities of DNA have to be injected

periodically.

2) LIPOPLEXES:

– Lipid DNA complexes; DNA construct surrounded by artificial

lipid layer.

– Most of it gets degraded by lysosomes.

 NON VIRAL VECTORS

3) DNA MOLECULAR CONJUGATES:

– Commonly used synthetic conjugate is
poly- L- lysine bound to specific target cell
receptor.
– Therapeutic DNA is then made to combine with
the conjugate to form a complex.
– It avoids lysosomal breakdown of DNA.

4) HUMAN ARTIFICIAL CHROMOSOME:

– Can carry a large DNA ie, with one or more
therapeutic genes with regulatory elements.
 METHODS OF GENE DELIVERY
PHYSICAL METHODS:
 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.
 METHODS OF GENE DELIVERY
CHEMICAL METHODS:
 USING DETERGENT MIXTURES
– Certain charged chemical compounds like Calcium phosphates are
mixed with functional cDNA 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 cDNA to pass through the cell.

 LIPOFECTION
– It is a technique used to inject genetic materials into a cell by means of
liposomes.

– Liposomes are artificial phospholipid vesicles used to deliver a

variety of molecules including DNA into the cells.
 Major Development

First Approved Gene Therapy .

Ashanthi De Silva - A rare
genetic disease called severe
combined immunodeficiency
(SCID).
Defective adenosine deaminase
gene results in deficiency of ADA
protein .
It plays important role in
deamination reaction.
Dr. W. French
Lack of healthy immune system .
Anderson with four-
year old Ashanthi De
Silva at U.S. National
Institutes of Health
Gene therapy for Lou Gehrig disease
 Abnormal level of VE growth factors affect the
capilary density in the spinal chord,infection
caused by this disease know as Lou Gehrig
disease or amytrophic lateral sclerosis.
 This disease was named after the popular bass
ball player Lou gerig.this disease is considere as a
gentical disorders.
Gene therapy for sicle cell anemia
CFTR immunohistochemical staining of nasal brushing cells
Gene therapy for cystic fibrosis
Genetically inherited disease
 Gene therapy for Hungtinton disease

 It is a genetical disease which destroys the

brain cell of brain.
 This disease was first caught in popular
american folk singer Woody Guthrine who
wrote ‘this land is your land’ song.
 Gene Therapy May Switch off
Huntington Disease

 RNA interference or gene silencing may be

a new way to treat Huntington disease.
 siRNA is designed to match the RNA copied
from a faulty gene
 HD results from polyglutamine repeat
expansion (CAG codon) in exon of
huntington gene
 Toxic gain of function on the huntington
protein .
 Gene Therapy for Parkinson's disease

 Liposomes coated in a polymer call

polyethylene glycol (PEG)
 Viral vectors are too big to get across the
"blood-brain barrier" This method has
potential for treating Parkinson's disease
Loss of dopaminergic neurons
 Tyrosine hydroxylase gene therapy
 Episomal based gene therapy
 Gene Therapy for Advanced Melanoma

 Reengineer lymphocytes for expression of TCR to

target and attack cancer cells in patients with
advanced metastatic melanoma
 Retroviral vector was constructed encoding the
pmel-1 TCR genes targeting the B16 melanoma
antigen, gp100
 Adoptive cell transfer
 Transduction of C57BL/6 lymphocytes resulted in
efficient pmel-1 TCR expression
 Gene therapy for Familial Hypercholesterolemia
 It is considered as a rare genetic disease.
 This disease typically seem in liver cells because of
having abnormal LDL receptor gene. They are
separated and cultured into billion of liver cells in a
lab.
 A retroviral vector is used to transfer the gene for LDL
receptor to culture liver cells.
 The cultured liver cells now have normal LDL
receptors.
 The cultured liver cells are injected into a vein that
leads to the liver. Liver cells begin to make the LDL
receptor and the patients level of harmful cholesterol
drops.
 Gene therapy for HIV infection

 Hematopeitic stem cell based gene therapy

against HIV infection has emerged as a
promising approach for the treatment of
HIV/AIDS.
 HIV -1 attachment to susceptible cells involve
binding of gp 120 to CD4 receptor and a
subsequently to a chemokine co-receptor,either
CCR5 or CXCR4.
 Studies have shown that a 32 bp deletion in
CCR5 generates a nonfuntional gene product.
 ADVANTAGES

 Gene therapy has the potential to eliminate and

prevent hereditary diseases such as cystic fibrosis,
ADA- SCID etc.

 It is a possible cure for heart disease, AIDS and

cancer.

 It gives someone born with a genetic disease a

chance to life.

 It can be used to eradicate diseases from the future

generations.
 Disadvantages
 Short-lived nature of gene therapy
 Immune response and toxicity
 Problems with viral vectors
 Restricted targeting of specific cell types
 Multigene disorders
 Insertional mutagenesis
 Religious concern
 Deaths may occured
 ETHICAL ISSUES

 Who will have access to therapy?

 Is it interfering with God’s plan?

 Should people be allowed to use gene

therapy to enhance basic human traits
such as height, intelligence etc.?

 Is it alright to use the therapy in the

prenatal stage of development in
babies?
 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

 Gene therapy have the potential to revolutionize the

practice of medicine.
THANK YOU!!!