SHRI RAM GLOBALSCHOOL

BIOLOGY INVESTIGATORY PROJECT

TOPIC: Gene Therapy

Submitted By
Rachit Jaiswal
Grade: XII
 CERTIFICATE

This is to certify that Master Rachit Jaiswal of Grade XII,

Shri Ram Global School has completed her project for
CBSE practical examination for the academic year 2023-24
under my supervision. He has taken great care and shown
utmost sincerity in the completion of the project. I further
certify that this project is upto my expectation and is as per
CBSE requirements.

Internal Examiner External Examiner

Principal
 ACKNOWLEDGEMENT

I would like to express my sincere gratitude to my biology

teacher Mrs. Divya Devi for their vital support, guidance, and
encouragement without which this project would not have
come forth. I would also like to thank Mrs. Manjusha Tripathi,
our principal, for her continued support and to provide the
facilities required to execute this project. Last, but not least, I
would like to thank my parents for their undivided support and
interest which always inspires me.

Rachit Jaiswal
 INDEX

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S.NO. CONTENT

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 BIBLIOGRAPHY
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 Study On Gene Therapy

Published on Mar 20, 2020

Abstract
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.
Based on the cause diseases can be broadly classified as:
Infections
These are diseases caused due to invasion of a foreign
parasitic organism. They are temporary because the
immune system of organisms can fight such pathogens
(disease causing organisms) to a certain extent hence
helping in prevention of the disease. The immune system
can also be aided with the use of several drugs. Apart
from easy treatment they can also be easily prevented
Lifestyle Diseases
Lifestyle diseases (also sometimes called diseases of
longevity or diseases of civilization interchangeably) are
diseases that appear to increase in frequency as countries
become more industrialized and people live longer. They
can include Alzheimer's disease, asthma, and obesity. Diet
and lifestyle are major factors thought to influence
susceptibility to many diseases. Drug abuse, tobacco
smoking, and alcohol drinking, as well as a lack of
exercise may also increase the risk of developing certain
diseases, especially later in life. These diseases can be
prevented completely by living a healthy lifestyle.

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.
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.

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.
Is It A Good Target For Gene Therapy?
To check this some questions must be answered:
Does the condition result from mutation? Yes.
Is the biology of the disorder known? Yes.
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.

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
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.

Reference
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