BIOLOGY

INVESTIGATORY
PROJECT
SESSION : 2024-25

SUBMITTED BY:
NAME : TOSI RIBA
CLASS : XII ‘B’ SCIENCE
ROLL NO. : 20

ACKNOWLEDGEME
NT
I take this opportunity to express my
gratitude towards my biology teacher
Jaishankar Sir for his precious and
valuable guidance which played a crucial
role in not only my investigatory project on
the topic “Gene Therapy”, but also
throughout the session with my studies. I
am very grateful to the school as well, for
giving me this opportunity. Last but not the
least I would like to thank my parents and
my friends for helping me in this project.
 CERTIFICATE

This is to certify that Tosi Riba

student of grade XII (12th) Science
‘B’ has successfully completed their
Biology Investigatory Project on “Gene
Therapy” under the guidance of the
Biology teacher, Jaishankar Sir.
Teacher’s External examiner’s
Principal’s
Signature Signature
Signature

SI NO. CONTENTS
1. Introduction
2. History & Types of Gene Therapy
4. Germline & Somatic Gene Therapy
6. Outcomes of Gene Therapy
7. Functional classification & Genetic

disorders
8. Gene Therapy
9. Targets for gene Therapy
10. Technologies used in Gene Therapy
11. Gene Targetting
12. Success cases of Gene Therapy
13. Advantages & Disadvantages
14. Challenges & Recent Upcoming
 15. Conclusion & Bibliography

INDEX

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 & 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 natients This studylarger
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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 Hutter has cured a man from HIV using
gene therapy.

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 fatal
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'tbehave 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

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.
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. 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.

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 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 Targetting
Gene delivery is one of the biggest challenges in the
field of gen 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.

Success Cases of gene therapy

1) Gene therapy cures blindness
Cure blindness of inherited condition. Leber's
conginetal amaurosis.
o Inherited disease caused by an abnormality in a
gene called RPE65.
 o 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 needlesafe 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.
 The gene instructs cells to begin making GABA
neurotransmitters to re-establish the normal
chemical balance that becomes dysfunctional as
the diseases progresses.

Advantages
1. Give a chance of normal life to baby born with
genetic disease.
 2. Give hope of healthy life to cancer patients.
3. For certain disease that do not have any cure
except gene therapy, it could save many lives.

Disadvantages
1. The genetic testing, screening and research in
finding the availability of certain gene is very
controversy.
2. May increase rate abortionif prenatal test regarding
baby with genetic disease is done.
3. The cost is very and the patient might need an
insurance to cover the treatment.
4. 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.

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.
4. 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.

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 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, Casg, to the DNA of interest. Caso can
cut the double strand, nick it, or even knock down
gene expression. After Caso 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.

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.

Reference
1. 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. https://en.wikipedia.org/wiki/Gene_therapy
5.
http://learn.genetics.utah.edu/content/tech/genetherap
y/
6. Handbook on therapy
http://ghr.nlm.nih.gov/handbook/therapy/
7. https://www.yourgenome.org/facts/what-is-gene-
therapy
8. https://knowgenetics.org/gene-therapy/
9. https://www.sciencedaily.com/terms/gene
therapy.htm