Gene Therapy 1

GENE THERAPY
“Technology always goes forward.

There are radical new technologies that
surprise us all the time.
And we’ve got a long time in the future to
go.
This is my conclusion:
Human evolution will be self-driven.”
Lee Silver, PhD, 3/98

Introduction
Genetic Counseling
Treating Genetic Disease
Removing an affected body part.
Replacing an affected body part or biochemical

with material from a donor.
Delivering pure, human proteins derived from

recombinant DNA technology to compensate for
the effects of a mutation.
Gene therapy, to replace mutant alleles.

Gene Therapy
 Use of DNA as a pharmaceutical agent
to treat disease.
 First conceptualized in 1972.
 Approved Gene Therapy experiment in
1990.
 The boy who lived in a bubble: Incredible images show
child who spent his entire life sealed from the outside
world in the desperate hope scientists would find a
cure for his auto-immune disease.
 David Vetter was born in September 1971 with a deadly

genetic disease.
 He was placed in a bubble due to Severe Combined

Immune Deficiency.
 David's elder brother, also David, died after eight

months due to the illness.
 Medics used David's experience to successfully treat

others with SCID
David was placed in his plastic bubble after he was born and remained
cocooned until he was 12.
Medics could only touch David using a special pair of gloves in a bubble designed
David Vetter, known as the Bubble Boy, spent 12 years living inside a
hermetically-sealed cocoon
Severe Combined Immunodeficiency
Disease (SCID)
oDavid Vetter, the “Boy in the Bubble”, received bone marrow

from his sister  unfortunately he died from a form of blood
cancer.
oSCID is caused by an Adenosine Deaminase Deficiency
(ADA).
oGene is located on chromosome 22 (32 Kbp, 12 exons).
oDeficiency results in failure to develop functional T and B

lymphocytes.
o September 14, 1990 at
NIH, French Anderson
and R. Michael Blaese
perform the first gene
therapy Trial.
o Ashanti (4 year old girl)
Her lymphocytes were
gene-altered (~109) ex
vivo
 retrovirus vector used
as a vehicle for gene
introduction using to carry
ADA gene (billions of
retroviruses used)
TYPES OF GENE THERAPY
Gene therapy
In vivo Ex vivo
Gene therapy approaches vary in invasiveness
Ex vivo gene therapy:

Cells can be altered outside
the body and then infused.
In situ gene therapy:
The functional gene plus the
DNA that delivers it (the
vector) are injected into a
very localized and accessible
body part, such as a single
melanoma skin cancer.
In vivo gene therapy:
(in the living body)
In the most invasive
approach, the gene and
vector are introduced directly
into the body.
in vivo and ex vivo schemes
EX VIVO
IN VIVO
http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm
In humans
Cancer 69%
General concerns
The Food and Drug Administration (FDA) has not yet approved
any human gene therapy product for sale.
Four major problems with gene therapy:

1) Short-lived nature of gene therapy. Very hard to achieve any long-term
benefits without integration and even with it.
2) Immune response. It reduces gene therapy effectiveness and

makes repetitive rounds of gene therapy useless
3) Problems with viral vectors . Toxicity, immune and inflammatory
responses, also fears that viral vector may recover disease-causing ability
4) Multigene disorders. Most commonly occurring disorders,
such as heart disease, Alzheimer's disease, arthritis, and diabetes,
are caused by the combined effects of variations in many genes.
Methods of gene delivery
(therapeutic constructs)
-- Injection of naked DNA into tumor by simple needle and syringe
-- DNA coated on the surface of gold pellets

which are air-propelled into the epidermis
(gene-gun), mainly non applicable to cancer
-- DNA transfer by liposomes

(delivered by the intravascular, intratracheal,
intraperitoneal or intracolonic routes)
-- Biological vehicles (vectors) such as viruses and bacteria.

Viruses are genetically engineered
so as not to replicate once inside the host.
They are currently the most efficient means of gene transfer.
In vivo techniques usually utilize viral vectors
Virus : carrier of desired gene
Virus is usually “crippled” to disable its ability to

cause disease.
Viral methods have proved to be the most
efficient to date.
Many viral vectors can stable integrate the
desired gene into the target cell’s genome.
Delivering methods of a
Gene
Physical Methods
Microinjection.
Direct DNA injection.
Gene gun.
Electroporation.
Chemical Methods
Receptor mediated gene delivery.
Embryo therapy through IVF-technology.
Biological Methods
Retrovirus.
Adeno-associated virus.
Adeno virus.
MOST COMMON VIRAL VECTORS
Retroviruses
can create double-stranded DNA copies of their RNA genomes. Can integrate into genome.
HIV, MoMuLV, v-src, Rous sarcoma virus
Adenoviruses
dsDNA viruses that cause respiratory, intestinal, and eye infections
in humans. Virus for common cold
Adeno-associated viruses
ssDNA viruses that can insert their genetic material
at a specific site on chromosome 19
Herpes simplex viruses

dsDNA viruses that infect a neurons. Cold sores virus
Bioethical
 Does the participant in a gene therapy trial truly understand
the risks?
 If a gene therapy is effective, how will recipients be selected,

assuming it is expensive at first?
 Should rare or more common disorders be the focus of gene

therapy research and clinical trials?
 What effect should deaths among volunteers have on

research efforts?
 Should clinical trials be halted if the delivered gene enters the

germline?

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GENE THERAPY

“Technology always goes forward.

Lee Silver, PhD, 3/98

Removing an affected body part.

Replacing an affected body part or biochemical

Delivering pure, human proteins derived from

Gene therapy, to replace mutant alleles.

 David Vetter was born in September 1971 with a deadly

 He was placed in a bubble due to Severe Combined

 David's elder brother, also David, died after eight

 Medics used David's experience to successfully treat

oDavid Vetter, the “Boy in the Bubble”, received bone marrow

oDeficiency results in failure to develop functional T and B

Ex vivo gene therapy:

Four major problems with gene therapy:

2) Immune response. It reduces gene therapy effectiveness and

-- DNA coated on the surface of gold pellets

-- DNA transfer by liposomes

-- Biological vehicles (vectors) such as viruses and bacteria.

Virus : carrier of desired gene

Virus is usually “crippled” to disable its ability to

Herpes simplex viruses

 If a gene therapy is effective, how will recipients be selected,

 Should rare or more common disorders be the focus of gene

 What effect should deaths among volunteers have on

 Should clinical trials be halted if the delivered gene enters the

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