Table of Contents

Introduction
Gene Therapy
Targets
Isolation of gene
Gene Targeting
Gene Delivery
Case Study Cystic Fibrosis
The Disease
Is it a good Target
Choosing Vectors
History
Challenges
Ethical Issues
Recent Upcoming
CRISPR
Conclusion
Bibliography
Websites
Books
Introduction
Diseases
The term disease broadly refers to any condition that impairs normal function, and is
therefore associated with dysfunction of normal homeostasis. When the functioning
of one or more organs or systems of the body is adversely affected, characterised by
various signs and symptoms, we say that we are not healthy, i.e., we have a
disease.

Health can be defined as a state of complete physical, mental and social well-being.
When people are healthy, they are more efficient at work. This increases productivity
and brings economic prosperity. Health also increases longevity of people and
reduces infant and maternal mortality.

Genetic Disorders
A genetic disorder is an illness caused by one or more abnormalities in the
genome, especially a condition that is present from birth (congenital). They are
medical disorders related to gene mutation.

Genetic disorders are heritable, and are passed down from the parents' genes. Other
defects may be caused by new mutations or changes to the DNA. In such cases, the
defect will only be heritable if it occurs in the germ line.
The same disease, such as some forms of cancer,
may be caused by an inherited genetic condition in
some people, by new mutations in other people, and
by non-genetic causes in still other people.

These diseases are totally random and difficult to

prevent as they are not caused by external agents.
Also as their root cause lies in the genome of the
organism their cure was thought to be impossible until the breakthrough research
unlocking the secrets of DNA leading to the development of biotechnology and
hence gene therapy.
Gene Therapy
We can think of a medical condition or
illness as a "broken window." Many
medical conditions result from flaws, or
mutations, in one or more of a person's
genes. Mutations cause the protein
encoded by that gene to malfunction.
When a protein malfunctions, cells that rely
on that protein's function can't behave
normally, causing problems for whole
tissues or organs. Medical conditions
related to gene mutations are called
genetic disorders.

So, if a flawed gene caused our "broken window," can we "fix" it? What are our
options?

1. Stay silent: ignore the genetic disorder and nothing gets fixed.

2. 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.

3. Put in a normal, functioning copy of the gene: if you can do this, it may solve
the problem!

If it is successful, gene therapy provides a way to fix a problem at its source. Adding
a corrected copy of the gene may help the affected cells, tissues and organs work
properly. Gene therapy differs from traditional drug-based approaches, which may
treat the problem, but which do not repair the underlying genetic flaw.

Targets for Gene Therapy

But now a question arises, which disorders or diseases can we target using gene
therapy? Many disorders or medical conditions might be treated using gene therapy,
but others may not be suitable for this approach. For a disease to be targeted by
gene therapy it must satisfy the following conditions:

1. The condition must result from mutations in one or more genes

2. To treat a genetic flaw, the knowledge of which gene(s) to pursue is

absolutely necessary. Also a DNA copy of that gene available in the
laboratory. The best candidates for gene therapy are the so-called "single-
gene" disorders - which are caused by mutations in only one gene.
 3. To design the best possible approach, knowledge about how the gene
factors into the disorder is required. For example:

Which tissues are affected?

What role does the protein encoded by the gene play within the cells of that
tissue?

Exactly how do mutations in the gene affect the protein's function?

4. Adding a normal copy of the gene should fix the problem in the affected
tissue. This may seem like obvious, but it's not. What if the mutated gene
encodes a protein that prevents the normal protein from doing its job? Mutated
genes that function this way are called dominant negative and adding back the
normal protein won't fix the problem.

5. The gene delivery to cells of the affected tissue must be possible. It

depends on:

How accessible is the tissue? Is it fairly easy (skin, blood or lungs), or more
difficult to reach (internal organs)?

What is the best mode of delivery?

The techniques of biotechnology have made it possible to isolate the required gene
in the laboratory and also deliver the gene.

Isolation of Gene of Interest

The first step is to find and isolate the gene that will be inserted into the genetically
modified organism. Finding the right gene to insert usually draws on years of
scientific research into the identity and function of useful genes. Once that is known
the DNA needs to be cut at specific locations to isolate the gene of interest. This can
be done by using restriction enzymes also known as molecular scissors which cut
DNA at specific sites containing palindromic DNA sequences. But in order to cut the
DNA with restriction enzymes, it needs to be in pure form, free from other macro-
molecules.

Isolation of DNA
Since the DNA is enclosed within the membranes, we have to break the cell open to
release DNA along with other macromolecules such as
RNA, proteins, polysaccharides and also lipids. This can
be achieved by treating the bacterial cells/plant or animal
tissue with enzymes such as lysozyme (bacteria),
cellulase (plant cells), chitinase (fungus). Genes are
located on long molecules of DNA intertwined with
proteins such as histones. The RNA can be removed by treatment with ribonuclease
whereas proteins can be removed by treatment with protease. Other molecules can
be removed by appropriate treatments and purified DNA ultimately precipitates out
after the addition of chilled ethanol. This can be seen as collection of fine threads in
the suspension.

Cutting of DNA

Restriction enzyme digestions are performed by

incubating purified DNA molecules with the
restriction enzyme, at the optimal conditions for
that specific enzyme. The cutting of DNA by
restriction endonucleases results in the fragments
of DNA. These fragments can be separated by a
technique known as gel electrophoresis. Since
DNA fragments are negatively charged molecules
they can be separated by forcing them to move
towards the anode under an electric field through
a medium/matrix. The separated bands of DNA
are analysed for the required gene and then it is cut out from the agarose gel and
extracted from the gel piece. This step is known as elution.

Multiplication of Gene (PCR)

PCR or polymerase chain reaction is then used to create multiple copies of the gene
of interest. In this reaction, multiple copies of the gene (or DNA) of interest is
synthesised in vitro using two sets of primers (small chemically synthesised
oligonucleotides that are complementary to the regions of DNA) and the enzyme
DNA polymerase. The enzyme extends the primers using the nucleotides provided in
the reaction and the genomic DNA as template. If the process of replication of DNA
is repeated many times, the segment of DNA can be amplified to approximately
billion times, i.e., 1 billion copies are made.

Gene Targeting
Gene delivery is one of the biggest challenges in the field of gene therapy.

Gene Delivery includes:

1. TARGETING the right cells.

2. ACTIVATING the gene. A gene's journey is not over when it enters the cell. It
must go to the cell's nucleus and be "turned on," meaning that its transcription and
translation are activated to produce the protein product encoded by the gene. For
gene delivery to be successful, the protein that is produced must function properly.

3. INTEGRATING the gene in the cells. The gene must stay put and continue
working in the target cells. If so, it must be ensured that the gene integrates into, or
becomes part of the host cell's genetic material, or that the gene finds another way to
survive in the nucleus without being rejected.

4. AVOIDING harmful side effects. Anytime an unfamiliar biological substance is

introduced into the body, there is a risk that it will be toxic or that the body will mount
an immune response against it. If the body develops immunity against a specific
gene delivery vehicle, future rounds of the therapy will be ineffective.

Choosing the Best Vector

There is no "perfect vector" that can treat every disorder. Like any type of medical
treatment, a gene therapy vector must be customized to address the unique features
of the disorder. We have learnt the lesson, of transferring genes into plants and
animals from bacteria and viruses, which have known this for ages how to deliver
genes to transform eukaryotic cells and force them to do what the bacteria or viruses
want.

Part of the challenge in gene therapy is choosing the most suitable vector for treating
the disorder. Some vectors commonly used are:

Viruses

Usually when we think of viruses, we think of them causing diseases such as the
common cold, the flu, and HIV/AIDS. When faced with the problem of gene delivery,
scientists looked to viruses. Why reinvent the wheel if there's a perfectly good one
out there? If we can modify viruses to deliver genes without making people sick, we
may have a good set of gene therapy tools.

Non-Viral Vectors

Although viruses can effectively deliver genetic material into a patient's cells, they do
have some limitations. It is sometimes more efficient to deliver a gene using a non-
viral vector, which has fewer size constraints and which won't generate an immune
response.

Non-viral vectors are typically circular DNA molecules, also known as plasmids. In
nature, bacteria use plasmids to transfer genes from cell to cell.
Scientists use bacteria and plasmids to easily and efficiently store and replicate
genes of interest from any organism.

Vectors used at present, are engineered in such a way that they help easy linking of
foreign DNA and selection of recombinants from non-recombinants.

These are not the only way to introduce alien DNA into host cells.
In a method known as micro-injection, recombinant DNA is directly injected into the
nucleus of an animal cell. In another method, suitable for plants, cells are
bombarded with high velocity micro-particles of gold or tungsten coated with DNA in
a method known as biolistics or gene gun.

Case Study
Cystic Fibrosis

The Disease A Genetic Disorder

Cystic fibrosis (CF), also known as mucoviscidosis,
is an autosomal recessive genetic disorder that
affects most critically the lungs, and also the
pancreas, liver, and intestine. It is characterized by
abnormal transport of chloride and sodium across
an epithelium, leading to thick, viscous secretions,
preventing the cilia from clearing debris which cause
symptoms such as coughing, poor digestion and
increased vulnerability to infection.

CF is caused by a mutation in the gene for the

protein cystic fibrosis transmembrane conductance
regulator (CFTR) gene on chromosome 7. Most
commonly, the mutation in the CFTR gene is a
three-base-pair deletion. This protein is required to
regulate the components of sweat, digestive fluids,
and mucus. CFTR
regulates the
movement of chloride
and sodium ions
across epithelial
membranes, such as the alveolar epithelia located in the lungs. Since all of the cells
of a CF patient have the defective protein, large quantities of thick, sticky mucus
build up throughout the lungs and other organs. This results in the severity of
symptoms seen in CF patients.

Challenges
Some the factors that have kept gene therapy from becoming an effective treatment
for genetic diseases are:

Short-lived nature of gene therapy - Before gene therapy can become a

permanent cure for any condition, the therapeutic DNA introduced into target
cells must remain functional and the cells containing the therapeutic DNA
must be long-lived and stable. Problems with integrating therapeutic DNA into
the genome and the rapidly dividing nature of many cells prevent gene
therapy from achieving any long-term benefits. Patients will have to undergo
multiple rounds of gene therapy.

Immune response -Anytime a foreign object is introduced into human

tissues, the immune system is designed to attack the invader. The risk of
stimulating the immune system in a way that reduces gene therapy
effectiveness is always a potential risk. Furthermore, the immune system's
enhanced response to invaders it has seen before makes it difficult for gene
therapy to be repeated in patients.

Problems with viral vectors - Viruses, while the carrier of choice in most
gene therapy studies, present a variety of potential problems to the patient --
toxicity, immune and inflammatory responses, and gene control and targeting
issues. In addition, there is always the fear that the viral vector, once inside
the patient, may recover its ability to cause disease.

Multigene disorders -Conditions or disorders that arise from mutations in a

single gene are the best candidates for gene therapy. Unfortunately, some the
most commonly occurring disorders, such as heart disease, high blood
pressure, Alzheimer's disease, 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.
Conclusion
Although early clinical failures led many to dismiss gene therapy as over-hyped,
clinical successes since 2006 have bolstered new optimism in the promise of gene
therapy. These include successful treatment of patients with the retinal
disease Leber's congenital amaurosis, X-linked SCID, ADA-SCID,
adrenoleukodystrophy, chronic lymphocytic leukaemia (CLL),acute lymphocytic
leukaemia (ALL),multiple myeloma, haemophilia and Parkinson's disease. These
recent clinical successes have led to a renewed interest in gene therapy, with
several articles in scientific and popular publications calling for continued investment
in the field.
Bibliography

Websites
http://en.wikipedia.org/wiki/Gene_therapy
http://www.trip2medi.com/treatmentCGeneTherapy.php
http://learn.genetics.utah.edu/content/tech/genetherapy/
http://ghr.nlm.nih.gov/handbook/therapy/
http://cystic-fibrosis.emedtv.com/cystic-fibrosis/cystic-fibrosis-gene-therapy.html
http://en.wikipedia.org

Books
12th NCERT Biology
Stryer Biochemistry