IDEAL INDIAN SCHOOL

BIOLOGY INVESTOGATORY
PROJECT

STUDY ON GENE THERAPY

NAME: HAJIRA NYSA

CLASS: 12 E
ROLL NO:
IDEAL INDIAN SCHOOL,DOHA-QATAR
P.O.BOX:2836
DOHA-QATAR
PHYSICS PROJECT WORK
DONE AND SUBMITTED BY:
HAJIRA NYSA
IN PARTIAL FULFILMENT OF REQUIREMENTS FOR
CLASS XII COURSE 2022-2023
SUPERVISED BY:
MRS.SHYMA
TEACHER IN CHARGE
IDEAL INDIAN SCHOOL
DOHA QATAR

SCHOOL SEAL
 IDEAL INDIAN SCHOOL,DOHA-QATAR
BONAFIDE CERTIFICATE
This is to certify that Hajira Nysa of class XII E
has done the Biology project work during the
academic year 2022-2023 at Ideal Indian
School,Doha Qatar.
Her examination roll number is:
Name of PGT:
Date: Signature:
Principal Signature:
Submitter to All-Indian Senior School Certificate
Examination in physics(Practical)conducted at
Ideal Indian School

Internal Examiner External Examiner

Name:_____________________ Name:_____________________
Signature:__________________ Signature:__________________
School:_____________________ School:_____________________
 ACKNOWLEDGMENT

First and foremost, I would like to express my sincere

gratitude to the Almighty for providing me with the
right resources, and ideas strength in order to help me
accomplish this project.
Secondly I would like to thank my biology teacher
Mrs.Shyma for the vital support and genuine guidance
throughout without which this project would not have
been successful.
Last but not least I would like to thank my family and
friends for their constant support and encouragement
in the completion of this project
S.NO CONTENTS

1 Objective
2 Introduction
3 History & development of gene therapy
4 Types of gene therapy
5 Outcome of gene therapy
6 Functional classification
7 Genetic disorder
8 Gene therapy
9 Targets of gene therapy
1o Techniques used in gene therapy
11 Gene targeting
12 Choosing the best vector
13 Challenges
14 Recent upcoming
15 Conclusion
16 Bibliography
 OBJECTIVE

Gene therapy is used to correct defective genes in order to

cure a disease or help your body better fight disease.
Researchers are investigating several ways to do this,
including: Replacing mutated genes. Some cells become
diseased because certain genes work incorrectly or no longer
work at all.
 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
 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 messenger RNA
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 setback to 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
✓ Color blindness
✓ Adrenoleukodystrophy
2011: Medical community accepted that it can cure HIV as in
2008, Gero Hutter has cured a man from HIV using gene therapy.
 TYPES OF GENE THERAPY
Germ Line gene therapy
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.

Eg: Genes introduced into eggs and sperms.

Somatic Cell Gene Therapy

Therapeutic genes transfered 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,

Eg: 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
 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
 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
 TARGETS OF 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?

.
 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 (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 synthesized in vitro using two sets of primers (small
chemically synthesized 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 TARGETTING

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.

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.
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
 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.
 RECENT UPCOMING

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.

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.
"It may also depend on the stage one attempts to carry out the
therapy, in terms of the patient’s age and level of advancement of
the disease," says Ramesar.
 CONCLUSIONS

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
1. Wikipedia
2. Science daily
3. http://en.wikipedia.org/wiki/Gene_therapy
4. http://www.trip2medi.com/treatmentCGeneTherapy.php
5. http://learn.genetics.utah.edu/content/tech/genetherapy/
6. http://ghr.nlm.nih.gov/handbook/therapy/
7. http://cystic-fibrosis.emedtv.com/cystic-fibrosis/cystic-
fibrosis-gene-therapy.html
8. http://en.wikipedia.org