ABSTRACT Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of diseases. It is a technique for correcting defective genes responsible for disease development. The first approved gene therapy experiment occurred on September 14, 1990 in US when Ashanti DeSilva was treated for ADA-SCID. Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

1. AN INVESTIGATORY PROJECT
on
“Gene Therapy’’
Name – Zoha Sajjad
Class – XII
Rollno. – 029
Subject – Biology
Kendriya Vidyalaya Gole Market, New Delhi-110001

2. Contents
i. Abstract
ii. Introduction
iii. History of gene therapy
iv. Types of gene therapy
v. Somatic gene therapy
vi. Germ line gene therapy
vii. Outcomes of gene therapy
viii. Functional classification
ix. Genetic disorders
Targets for gene therapy
Technologies used in gene therapy
x. Gene Targeting
xi. Success cases of gene therapy
xii. Advantages & disadvantages
xiii. Challenges
xiv. Recent upcoming
xv. Conclusion
xvi. References

3. ABSTRACT
Gene therapy is the introduction of genes into existing cells to
prevent or cure a wide range of diseases. It is a technique for
correcting defective genes responsible for disease development.
The first approved gene therapy experiment occurred on
September 14, 1990 in US when Ashanti DeSilva was treated for
ADA-SCID. Gene therapy is designed to introduce genetic
material into cells to compensate for abnormal genes or to make
a beneficial protein. If a mutated gene causes a necessary protein
to be faulty or missing, gene therapy may be able to introduce a
normal copy of the gene to restore the function of the protein. A
gene that is inserted directly into a cell usually does not function.
Instead, a carrier called a vector is genetically engineered to
deliver the gene. Certain viruses are often used as vectors
because they can deliver the new gene by infecting the cell. The
viruses are modified so they can't cause disease when used in
people. Some types of virus, such as retroviruses, integrate their
genetic material (including the new gene) into a chromosome in
the human cell. Other viruses, such as adenoviruses, introduce
their DNA into the nucleus of the cell, but the DNA is not
integrated into a chromosome.

4. Acknowledgment
I would like to express my special thanks of gratitude to my
teacher (name) as well as our principal (Name) who gave me
the golden opportunity to do this wonderful project on the topic
“Gene therapy”, which also helped me in doing lot of literature
study and research and I came to know many new thing.
Secondly I would also like to thank my parents and friends who
helped me a lot in finalizing this project within the limited time
frame.
Zoha Sajjad

5. INTRODUCTION
Gene therapy is an experimental technique that uses genes to treat or
prevent disease. In the future, this technique may allow doctors to treat a
disorder by inserting a gene into a patient’s cells instead of using drugs or
surgery. Researchers are testing several approaches to gene therapy,
including:
 Replacing a mutated gene that causes disease with a healthy copy of
the gene.
 Inactivating, or “knocking out,” a mutated gene that is functioning
improperly.
 Introducing a new gene into the body to help fight a disease.
Although gene therapy is a promising treatment option for a number of
diseases (including inherited disorders, some types of cancer, and certain
viral infections), the technique remains risky and is still under study to
make sure that it will be safe and effective. Gene therapy is currently
being tested only for diseases that have no other cures. An experimental
technique for correcting defective genes that are responsible for disease
development.
The most common form of gene therapy involves inserting a normal gene
to replace an abnormal gene. It is intracellular delivery of genes to
generate a therapeutic effect by correcting an existing abnormality.

6. History and Development of Gene Therapy
1960 : The concepts of gene therapy was introduced.
1970: Friedmann and Roblin author of paper in science tittled “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 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 messengerRNA derived
from defective gene.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 gene intended to
correct hereditary diseases.
1999: Death of Jesse Gelsinger in a gene therapy experiment resulted in a
significant setbackto gene therapy research in the United States.
2006: Scientists at the National Institute 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 disase
 Colour blindness
 Adrenoleukodystrophy
2011: Medical community accepted that it can cure HIV as in 2008,
Gero Hutterhas cured a man from HIV using gene therapy.

7. TYPES OF GENE THERAPY
1) Germ Line gene therapy
2) Somatic gene therapy
Germ line gene therapy
Therapeutic genes transferred into the germ cells. It is heritable and
passed on to later generations. Result is permanent changes. Potential for
offering a permanent therapeutic effect for all who inherit the target gene.
Possibility of eliminating some diseases from a particular family.
E.g. Genes introduced into eggs and sperms.
Somatic Cell Gene Therapy
Therapeutic genes transferred into the somatic cells. Affects only the
target cells in the patient, and is not passed to future generations.
Short-lived because the cells of most tissues ultimately die and are
replaced by new cells. Transporting the gene to the target cells or tissue is
also problematic.
E.g. Introduction of genes into bone marrow cells, blood cells, skin cells
etc.
Outcome of gene therapy
 Replaces a mutated gene with a healthy one.
 Deactivates a gene that isn’t functioning properly
 Introduces a new gene in the body to help fight the disease
 Enhances the effect of a normally functioning gene.
 Activates the gene that was shut down during fetal life.

8. Functional Classification
Based on the purpose of gene therapy it can be –
1) Gene replacement therapy
2) Gene deactivation therapy
3) Transgenesis
4) Gene Enhancement therapy
5) Gene activation therapy
GeneticDisorders
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'tbehave normally, causing problems for whole
tissues or organs. Medical conditions related to gene mutations are called
genetic disorders.

9. 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.
Targetsfor 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?

10.  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?
Techniques used in gene therapy
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

11. (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
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:

12. 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 BestVector
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

13. deliver genes without making people sick, we may have a good set of gene
therapy tools.
General advantages of viral vectors:
 They're very good at targeting and entering cells.
-Some viral vectors might be engineered to target specific types of cells.
-They can be modified so that they can't replicate and destroy the cell.
General drawbacks of viral vectors:
A virus can't "expand" to fit a piece of genetic material larger than it is
naturally built to carry. Therefore, some genes may be too big to fit into a
certain type of virus.
 Viruses can cause immune responses in patients, resulting in two
potential outcomes:
- Patients may get sick.
 A patient's immunity to a virus may prevent him from responding to
repeated treatments.
 However, modern viral vectors have been engineered without most of
the proteins that would cause an immune response
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.

14. 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.
Cystic Fibrosis
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.

15. Success Cases of gene therapy
1)Gene therapy cures blindness
Cure blindness of inherited condition.
Leber’s conginetal amaurosis
-Inherited disease caused by an abnormality in a gene
called RPE65.
-The condition appears at birth or in the first few months
of life and causes progressive worse and loss of vision.
How it work??
 Used harmless viruses
 Enable access to the cells beneath the retinas of patients.
 By using a very fine needle – safe in an extremely fragile
tissue and can improve vision in a condition previously
considered wholly untreatable.
2)Gene therapy reduces Parkinson’s Disease
Symptoms
 It significantly improved the weakness of the symptoms
such as tremors, motor skill problems, and rigidity.
 Main- overactive brain region : the subthalamic nucleus
should be introduced with gene.
 That would produce GABA-an inhibitory chemical- then
they could potentially quiet that brain region and alleviate
tremors.
How it works??
 Done with local anesthesia, used a harmless, inactive virus [ AAV-2
GAD]
 Deliver the GAD gene into the pateinet’s subthalamic nucleus.

16.  The gene instructs cells to begin making GABA neurotransmitters to
re-establish the normal chemical balance that becomes
dysfunctionalas the diseases progresses.
Advantages
 Give a chance of normal life to baby born with
genetic disease.
Give hope of healthy life to cancer patients.
For certain disease that do not have any cure except
gene therapy, it could save many lives.
Disadvantages
 The genetic testing, screening and research in findind the
availability of certain gene is very controversy.
 May increase rate abortionif prenatal test regarding baby
with genetic disease is done.
The cost is very and the patient might need an insurance to
cover the treatment.
Cosmetic industry may monopolized this gene therapy if it
is used in enhancing beauty and in vanishing the aging
effect, rather than used for treatment of a disease.

17. Is It A Good Target For Gene Therapy?
To check this some questions must be answered:-
1. Does the condition result from mutation?
Yes.
2. Is the biology of the disorder known?
Yes.
3. Will adding a normal copy of the gene fix the problem in the affected
tissue?
Yes. While the mutated CFTR gene encodes a non-functional ion channel
protein, it will not prevent a normal CFTR channel protein from working
properly. Therefore, adding a normal copy of the CFTR gene should fix
the problem
Is it feasible to deliver the gene to the cells of the affected tissue? Yes, in
part. Treating the lungs of patients with CF might be feasible, since the
lung surfaces are exposed to the air and somewhat easy to reach. Because
the digestive system is less accessible, however, it might be a more
difficult region to treat.
Hence we can conclude that it is a perfect disease to be treated by gene
therapy.

18. 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
anylong-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

19. such as these would be especially difficult to treat effectively using gene
therapy.
Recent Upcoming
CRISPR
CRISPR stands for clustered regularly interspaced short palindromic
repeats. These RNA sequences serve an immune function in archaea and
bacteria, but in the last year or so, scientists have seized upon them to
rewrite genes. The RNA sequence serves as a guide to target a DNA
sequence in, say, a zygote or a stem cell. The guide sequence leads an
enzyme, Cas9, to the DNA of interest. Cas9 can cut the double strand,
nick it, or even knock down gene expression. After Cas9 injures the DNA,
repair systems fix the sequence - or new sequences can be inserted.
CRISPR
It isn't the first or only method of gene repair therapy that’s been
developed, but the CRISPR technology, says Ramesar, is so special
because, unlike previous methods which were more laborious and could
only target one kind of cell in the body, it appears to be a "one size fits all
delivery", adaptable for different tissues. The procedure also seems
relatively simple to perform.
Exciting as the development may be, CRISPR won’t be delivering instant
cures just yet.
Ramesar says, from his initial impressions of the literature, that it would
seem that localised, accessible abnormal tissue (as in the retina or skin)
could be targeted more easily.
Conditions affecting the body more systemically, however, such as certain
developmental syndromes, or central nervous system disorders, might be
problematic in terms of getting the repair technology into enough of the
target cells in that tissue to make an effective difference.

20. 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 successfultreatment 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.

21. Reference
1. What is gene therapy https://ghr.nlm.nih.gov/primer/therapy/genetherapy
2. https://ghr.nlm.nih.gov/primer/therapy/genetherapy
3. Gene therapy information http://uniqure.com/patients/Gene-Therapy-Information.pdf
4. http://en.wikipedia.org/wiki/Gene_therapy
5. Gene Therapy treatment
http://www.trip2medi.com/treatmentCGeneTherapy.php
6. http://learn.genetics.utah.edu/content/tech/genetherapy/
7. Handbook on therapy http://ghr.nlm.nih.gov/handbook/therapy/
8. Cystic fibrosis
http://cystic-fibrosis.emedtv.com/cystic-fibrosis/cystic-fibrosis-gene-therapy.ht
ml
9. http://en.wikipedia.org
10. https://www.yourgenome.org/facts/what-is-gene-therapy
11. https://knowgenetics.org/gene-therapy/
12. https://www.sciencedaily.com/terms/gene_therapy.htm