GENE

THERAPY

By- AVNEET KAUR

.
MODERATOR- Prof .VIRENDRA SINGH
1

What is gene therapy ?

Imagine that you accidentally broke

one of your neighbor's windows

I.

II.
III.

Stay silent: no one will ever find out

that you are guilty, but the window
doesn't get fixed.
Repair it with some tape: not the best
long-term solution.
Put in a new window: not only do you
solve the problem, but also you do
the honorable thing.
3

Many medical conditions result from

flaws in one or more genes. What are
your treatment options?
I.
II.

III.

Stay silent: ignore the genetic disorder

and nothing gets fixed.
Try to treat the disorder with drugs or
other approaches: depending on the
disorder, treatment may or may not be a
good long-term solution.
Put in a normal, functioning copy of the
E solve the
gene: if you can do this,EitNmay
G
Y
P
problem!
A
ER

TH

What is Gene Therapy?

Gene therapy is the insertion of genes into an

individual's cells and tissues to treat a
disease, such as a hereditary disease in which
a deleterious mutant allele is replaced with a
functional one.

The technology is still in its infancy

THE PRINCIPLE
A normal gene inserted to compensate for a
nonfunctional gene.
An abnormal gene traded for a normal gene
An abnormal gene repaired through selective

reverse mutation.

Change the regulation of gene pairs

DNA, Chromosomes, &

Genes: A Brief Introduction

WHAT IS A CELL?

Basic building blocks of all living things.

Human body composed of trillions of

cells.

Cell contain bodys hereditary material.

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NUCLEUS
Cells command

center

Houses DNA

DNA: The Genetic Blueprint

Hereditary material in humans and almost all

other organisms.

The information is stored as a code (made up of

four chemical bases):
adenine (A), guanine (G), cytosine (C) and
thymine (T).

Consists of about 3 billion bases. The order, or

sequence, of these bases determines the
information
available
for
building
and
maintaining an organism.
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DNA bases pairs A

with T and C with G.

Each base attaches to

a sugar molecule and a
phosphate molecule.

Together,
a
base,
sugar, and phosphate
are called a nucleotide.

Nucleotide form a
spiral called a double
helix.
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What is a
chromosome?

Each chromosome
is made up of DNA
tightly coiled many
times around
proteins called
histones that
support its structure.

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What is a gene?

Genes are made up of DNA.

Each chromosome contains

many genes.

In humans, genes size- from a

few hundred DNA bases to
more than 2 million bases.

Human has estimated between

20,000 and 25,000 genes
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How Genes Work...?

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How do genes direct the

production of proteins?

genes contain the information needed to make

functional molecules called proteins.
journey from gene to protein consists of two major
steps:
transcription
and
translation.
TRANSCRIPTION

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What Are Genetic

Disorders?

Sickle Cell Disorder

Genetic disorder is a disease

caused by a "variation" or
"mutation of a gene.

Genetic disorders can be

passed on to family members
who
inherit
the
genetic
abnormality.

Most
disorders
involving
genetic factors, such as heart
disease and most cancers, arise
from a interplay of multiple
genetic
changes
and
environmental factors.
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Why Genetic Disorders?

When genes are

altered so that the
encoded proteins
are unable to carry
out their normal
functions, genetic
disorders can result.

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Gene therapy -Background

1990 - The first gene therapy journal

published,

Human

Gene

Therapy

1990 - The first approved gene therapy

clinical trial took place when Ashanthi
DeSilva, a 4 year old girl (Maryland )
with ADA-deficient Severe Combined
Immunodeficiency , was given her own T
cells engineered with a retroviral vector
carrying

normal

ADA

gene.
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2000

-The

first

gene

therapy

cure

was

reported

when

Alain

Fischer
succeeded

(Paris)
in

totally

correcting children with

SCID or bubble boy
syndrome

Bubble BOY
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How It Works ?
A vector delivers the therapeutic gene into a
patients target cell
The target cells become infected with the viral
vector
The vectors 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
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Steps in Gene
Therapy

http://encarta.msn.com/media_461561269/Gene_Therapy.html

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Somatic and Germ Line

Gene Therapy

Gene therapy can target somatic (body) or

germ (egg and sperm) cells.
In somatic gene therapy the recipient's
genome is changed, but the change is not
passed on to the next generation.
Germ line gene therapy the newly
introduced gene is passed on to the
offspring.
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TRANSFECTION
is the introduction of
DNA into animal or
plant cells

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Methods of Transfection
Method

Viral

Non-viral

Chemical

Physical

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Viral vectors
Viral

vectors use viral genome

to carry therapeutic gene(s) and
to infect human body cells
Adenovirus (common cold)
Adeno-Associated Virus
Retrovirus (HIV)
Herpes simplex virus (cold
sores)

Viruses must be engineered so that they can neither produce disease nor

spread (extremely effective at infecting human cells)

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VIRAL VECTORS

Gene Therapy Depends on Delivery of

Corrective Genes
Viral vectors are a tool commonly used by
molecular biologists to deliver genetic
material into cells. Viruses have evolved
specialized molecular mechanisms to
efficiently transport their genomes inside the
cells they infect.

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A new gene is injected into an adenovirus vector, which is

used to introduce the modified DNA into a human cell. If the
treatment is successful, the new gene will make a functional
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protein.

Limitation of Direct Gene

Induction

Simplest method
Limited approachused only with certain
tissues and requires
large amounts of
DNA.

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Non-viral
methods

Injection of Naked DNA

Physical Methods :

Electroporation
Gene Gun
Sonoporation
Magnetofection

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Chemical Methods:

Liposomes
Dendrimers

Hybrid methods:

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Injection of Naked
DNA

simplest method of non-viral transfection.

Clinical trials carried out of intramuscular injection

of a naked DNA plasmid occurred with some
success.

In addition trials with naked PCR product,

showed similar or greater success.

Cellular uptake of naked DNA is generally

inefficient.
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Physical Methods :
Electroporation:

Uses short pulses of high

voltage to carry DNA
across the cell membrane.

shock causes temporary

formation of pores in the
cell membrane, allowing
DNA molecules to pass
through.
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Efficient but high rate of cell death limited its

use, including clinical applications.
ELECTRON-AVALANCHE TRANSFECTIONnew method of electroporation
By using a high-voltage plasma discharge, DNA
was efficiently delivered following very short
(microsecond) pulses.
Efficient and causes less cellular damage.
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Gene Gun

DNA is coated with

gold particles and
loaded into a device
which generates a
force to achieve
penetration of
DNA/gold into the
cells.

35

Sonoporation

Application Of
Ultrasound that creates
transient pores in a cell
membrane, allowing drug
molecules, proteins, or
foreign DNA to enter the
cell.
36

Magnetofection

Principle : nucleic acids

are inserted with cationic
magnetic nanoparticles.

these molecular complexes

are then concentrated and
transported into cells
supported by an
appropriate magnetic
field.
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Chemical Methods:

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LIPOSOMES

small diameter,
hollow particles
made of lipid
molecules.

Packaged with
genes and injected
into tissues

39

Dendrimers
highly branched macromolecule with a spherical
shape.
Cationic dendrimer ( one with a positive surface
charge is constructed).
HOW IT WORKS
In presence of genetic material such as DNA or
RNA it do temporary association with the nucleic
acid.
On reaching its destination the dendrimer-nucleic
acid complex is then taken into the cell via
endocytosis

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Hybrid methods:

Virosomes : combine liposomes with an

inactivated HIV or influenza virus.

More efficient gene transfer in

respiratory epithelial cells than either
viral or liposomal methods alone.

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TYPES OF GENE THERAPY

1.Ex vivo gene therapy
2.In vivo gene therapy
3. Antisense therapy

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Ex vivo gene therapy

Cells from diseased
person are removed
Then, they are treated in
laboratory conditions.
Finally, they are
reintroduced to the
patient

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In vivo gene therapy

Introducing genes directly

into tissues or organs

without removing body
cells.
Challenge is delivery only
to intended tissues
Viruses act as vectors for
gene delivery, but some
injected directly into tissue

44

Antisense therapy

When the genetic sequence of a particular

gene is known to be causative of a particular
disease, a strand of nucleic acid (DNA, RNA
or a chemical analogue) is synthesized that
binds to the messenger RNA (mRNA)
produced by that gene and inactivates it,
effectively turning that gene "off".

45

Antisense Therapy

Antisense therapy
is not strictly a form
of gene therapy, but
is a geneticallymediated therapy
and is often
considered together
with other methods

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Problems with Gene

Therapy

Short Lived

Hard to rapidly integrate therapeutic DNA into

genome and rapidly dividing nature of cells
prevent gene therapy for 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
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Viral Vectors

Multigene Disorders

patient could have toxic, immune, inflammatory

response
also may cause disease once inside

Heart disease, high blood pressure,

Alzheimers, arthritis and diabetes are hard to
treat because you need to introduce more than
one gene

May induce a tumor if integrated in a tumor

suppressor gene because of insertional
mutagenesis.

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Current Status
FDA hasnt approved any human gene therapy
product for sale
REASONS:
In 1999, 18-year-old Jesse Gelsinger died from
multiple organ failure 4 days after treatment for
ornithine transcarboxylase deficiency.

Death was triggered by severe immune response to

adenovirus carrier

January 2003, halt to using retrovirus vectors in

blood stem cells because children developed
leukemia-like condition after successful treatment for
X-linked severe combined immunodeficiency
disease
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RECENT
DEVELOPMENTS
Genes get into brain using liposomes

coated in polymer,

polyethylene glycol
potential for treating Parkinsons disease

RNA interference or gene silencing to treat Huntingtons

abnormal protein wont be produced

Create tiny liposomes that can carry therapeutic DNA through

pores of nuclear membrane

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Unresolved Questions
Can gene expression be controlled in the
patient?
What happens if normal gene is
overexpressed?
How long will the therapy last?
What is the best vector to use?
What is the minimum number of cells
needed to infect to achieve success?
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References
1.TEXTBOOK OF MICROBIOLOGY BY
ANANTNARAYAN.
2.TEXTBOOK OF BIOCHEMISTRY BY
SATYANARAYAN.
3.TEXTBOOK OF BIOCHEMISTRY BY
HARPER.
4.GENETHERAPY HANDBOOK.

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THANK YOU.

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