GENE THERAPY

BY
B.E.N
13th JULY 2015

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Introduction
Classes of gene therapy
Strategies for gene therapy
Methods used for gene therapy
Factors that have kept gene therapy ineffective for
treatment of genetic diseases
Developments in gene therapy research
Ethical concerns

Introduction

Gene therapy is a technique that involves insertion of

normal genes to correct defective genes responsible
for disease development.

It is the use of DNA as a pharmaceutical agent to treat

disease.

It derives its name from the idea that DNA can be

used to supplement or alter genes within an
individual's cells as a therapy to treat disease

The most common form of gene therapy

involves using DNA that encodes a
functional, therapeutic gene in order to
replace a mutated gene.

Other forms involve directly correcting a

mutation or using DNA that encodes a
therapeutic protein (rather than a natural
human gene) to provide treatment.

Gene therapy was first conceptualized in

1972, with the authors urging caution before
commencing gene therapy studies in humans.

On September, 1990, the first gene therapy

was performed by Dr. William French Anderson
on a four-year-old girl, Ashanti DeSilva, at the
National Institutes of Health, Bethesda, A girl
was treated for ADA-SCID. Since then, over
1,700 clinical trials have been conducted
using a number of techniques for gene
therapy.

APPROACHES FOR CORRECTING FAULTY GENES

A normal gene may be inserted into a

non-specific
location
within
the
genome to replace a nonfunctional
gene. This approach is most common

An

abnormal gene could be swapped

for a normal gene through homologous
recombination.

 The

abnormal gene could be repaired

through selective reverse mutation,
which returns the gene to its normal
function.

The

regulation of a particular gene

could be altered (the degree to which
a gene is turned on or off)

A) Somatic Gene Therapy

In somatic gene therapy, the therapeutic genes are

transferred into the somatic cells or body of a
patient.

Any modifications and effects will be restricted to

the individual patient only and will not be inherited
by the patient's offspring or later generations.

Somatic gene therapy represents the mainstream

line of current basic and clinical research, where
mRNA is used to treat a disease in an individual.

In germ line gene therapy, Germ cells, i.e., sperm or eggs,

are modified by the introduction of functional genes, which
are integrated into their genomes.

This would allow the therapy to be heritable and passed on

to later generations (offsprings).

Although this should, in theory, be highly effective in

counteracting genetic disorders and hereditary diseases,
many jurisdictions prohibit this for application in human
beings for a variety of technical and ethical reasons

Pronuclear (PI) Injection

(3) Strategies for Gene Therapy

1. Ex vivo, which means outside the body
Cells from the patients blood or bone marrow are
removed and grown in the laboratory.

They are then exposed to a virus carrying the

desired gene. The virus enters the cells, and the
desired gene becomes part of the DNA of the cells.

The cells are allowed to grow in the laboratory

before being returned to the patient by injection into
a vein or bone marrow.

2. In vivo, which means inside the body

No cells are removed from the patients body.

Instead, vectors are used to deliver the desired
gene to cells in the patients body.
3. In Situ
Vector is placed directly into the affected tissues .

List of Common Genetic Disorders

Disorder

Mutation

Chromosome

DCP

15

Color blindness

Cystic fibrosis

Down syndrome

21

Haemophilia

Klinefelter's
syndrome

Angelman
syndrome

Two methods are involved

(i) Non-viral mediated gene delivery method
(ii) Viral mediated gene delivery method

(I) Non-viral mediated gene delivery method

(i)Direct injection of therapeutic DNA into target cells
This approach is limited in its application because it can
be used only with certain tissues and requires large
amounts of DNA
(ii) Liposomes
Creation of an artificial lipid sphere with an aqueous core
In this method, the liposome, which carries the
therapeutic DNA, is capable of passing the DNA
through the target cell's membrane

(iii) Inorganic nanoparticles.

Chemically linking the DNA to a molecule that will bind to special
cell receptors. Once bound to these receptors, the therapeutic
DNA constructs are engulfed by the cell membrane and passed
into the interior of the target cell
(iv) Researchers also are experimenting with introducing a
47th (artificial human) chromosome into target cells.
This chromosome would exist autonomously alongside the
standard 46, not affecting their workings or causing any
mutations.
A problem with this potential method is the difficulty in delivering
such a large molecule to the nucleus of a target cell.

There are other several methods for non-viral gene

therapy, including, electroporation, the gene gun,
sonoporation, magnetofection etc.

E.g In 2003 a University of California, Los Angeles research

team inserted genes into the brain using liposomes coated in a
polymer called polyethylene glycol. The transfer of genes into
the brain is a significant achievement because viral vectors are
too big to get across the blood-brain barrier. This method has
potential for treating Parkinson's disease.

Gene gun
OR
microprojectile
bombardment
biolistics
Tiny DNAcoated particles
are shot into
the cells

The Gene Gun

Helium chamber
Rupture disk

Macrocarrier
DNA coated
gold particle
Stopping screen

Focusing device

PDS1000 Microparticle Delivery System

Target tissue

This method employs viruses as vehicles to transport

the desired genes to the target cells
(These are recombinant viruses sometimes
called biological nanoparticles)
Different types of viruses are used as vectors
for gene therapy
Retroviruses
A class of viruses that can create double-stranded
DNA copies of their RNA genomes

Adenoviruses - A class of viruses with doublestranded DNA genomes that cause respiratory,
intestinal, and eye infections in humans

Adeno-associated viruses - A class of small, singlestranded DNA viruses that can insert their genetic
material at a specific site on chromosome 19

Herpes simplex viruses

A class of double-stranded DNA viruses that infect a
particular cell type, e.g. Neurons

In most gene therapy studies, a "normal" gene is

inserted into the genome to replace an "abnormal,"
disease-causing gene

A carrier molecule called a vector must be used to

deliver the therapeutic gene to the patient's target
cells

Currently, the most common vector is a virus that has

been genetically altered to carry normal human DNA

Scientists have manipulated the virus genome to

remove disease-causing genes and insert therapeutic
genes.

When target cells are infected with the viral vector.

The vector then unloads its genetic material
containing the therapeutic human gene

Then, generation of a functional protein product from

the therapeutic gene restores the target cell to a
normal state.

(i) Short-lived nature of gene therapy

- Problems with integrating therapeutic DNA into the
genome and the mortal nature of many cells prevent gene
therapy from achieving any long-term benefits. Patients
will have to undergo multiple rounds of gene therapy
(ii) Immune response
- Anytime a foreign object is introduced into human
tissues, the immune system is designed to attack the
invader

Furthermore, the immune system's enhanced

response to invaders it has seen before makes it
difficult for gene therapy to be repeated in patients
(iii) Problems with viral vectors
-Though viruses are the carriers of choice in most gene
therapy studies, there some possible potential
problems to the patient; toxicity, immune and
inflammatory responses and the fear that the viral
vector, once inside the patient, may recover its ability
to cause disease.

(iv) Multigene disorders

-Conditions or disorders that arise from
mutations in a single gene are the best
candidates for gene therapy
-Unfortunately, some of the most
commonly occurring disorders, such as
heart disease, HBP, arthritis, and
diabetes, are caused by the combined
effects of variations in many genes.
- Multigene or multifactorial disorders
such as these would be especially
difficult to treat effectively using gene
therapy.

If the DNA is integrated in the wrong place in the

genome, for example in a tumor suppressor gene, it
could induce a tumor

This has occurred in clinical trials for X-linked severe

combined immunodeficiency (X-SCID) patients, in
which hematopoietic stem cells were transduced with a
corrective transgene using a retrovirus and this led to
the development of T cell leukemia in 3 of 20 patients.

Since gene therapy is relatively new and at

an experimental stage, it is an expensive
treatment to undertake.

This explains why current studies are focused

on illnesses commonly found in developed
countries, where more people can afford to
pay for treatment.

It may take decades before developing

countries can take advantage of this
technology.

Scientists currently know the functions

of only a few genes. Hence, gene
therapy can address only some genes
that cause a particular disease.

Worse, it is not known exactly whether

genes have more than one function,
which creates uncertainty as to
whether replacing such genes is indeed
desirable.

Generally, Gene therapy is very

promising

Sickle cell was successfully treated in

mice. (2002)
Thalassaemia, cystic fibrosis and
some cancers were successfully
treated (2002).

Leber's congenital amaurosis, a type of inherited

childhood blindness caused by a single abnormal gene.
The procedure has already been successful at restoring
vision for dogs. This is the first trial to use gene therapy
in an operation to treat blindness in humans.

Gene Therapy cured deafness in guinea pigs (Feb

2005)- A gene, called Atoh1, which stimulates the hair
cells' growth, was delivered to the cochlea by an
adenovirus

"Bubble boy disease" more accurately called

severe combined immunodeficiency, or SCID
results from a malfunctioning gene in the bone
marrow, which produces immune cells.

Because the immune system is so severely

weakened, any infection or disease could result in
death. The children are therefore forced to live
very sheltered and isolated lives.

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children with bubble boy disease.

1 child did not respond to treatment
8 children cured
2 children came down with leukemia

THERAPY IN NEWS
Slimming Gene discovered
that regulates Body fat

The scientists therefore have hopes in

new medicines with which obesity
could be fought. Their research bas
been published in 'The EMBO Journal'
(doi: 10.1038/emboj.2009.305).

Some Questions to Consider

What is normal and what is a disability or disorder,
and who decides?

Are disabilities diseases? Do they need to be cured

or prevented?

Does searching for a cure demean the lives of

individuals presently affected by disabilities?

Ethical Considerations cont

Who should pay?
Preliminary attempts at gene therapy are exorbitantly
expensive. Who will have access to these therapies?
Who will pay for their use?
Gene therapy currently cost up to $200,000 per
treatment.
Unforeseen

consequences
Is it possible that eliminating defective genes from
our genome may have unforeseen consequences

 Religious

Objections
Majority of the world believes in some type of
higher power.

The majority of religions states that everyone

should be happy with what they have.

Do we have the right to play GOD?