BIOLOGY

INVESTIGATORY
PROJECT

Topic – Sickle Cell Anaemia

Name – Vyom Sharma
Class – 12th B
Roll No.- 12230
School – Kendriya Vidyalaya no.1
Sector 30
1
 Certificate

This is to certify that Vyom M

Sharma , a student of class 12th B
has completed the research on the
below mentioned project, under
the guidance of Mr. Shelender
Singh {biology teacher} during the
year 2023-2024 in partial
fulfilment of the biology practical
examination conducted by CBSE ,
Gandhinagar.

_____________________
Signature of class teacher
_____________________
Signature of Principal
 2

Acknowledgement

I would like to express a deep sense

of thanks and gratitude to my
biology teacher Mr.Shelender Singh
for guiding me immensely through
the course of my project. Their
constructive advice and constant
motivation have been responsible
for the successful completion of my
project. My sincere thanks to my
parents for their motivation and
support. I must thank my
classmates for their timely help and
support for compilation of this
project.
 3

INDEX
Content Page no.
Certificate 02
Acknowledgment 03
Topic 05
Abstract 06
Genetic Disorders 07
Gene Therapy 08
Targets of Gene Therapy 09
Isolation of Gene of 11
Interest
Gene Targeting 13
Choosing the Best Vector 14
Benefits of Gene Therapy 17
Challenges of Gene 19
 Therapy
Recent Advancements 21
Conclusion 22
References 23

TOPIC

Gene Therapy
Gene therapy is a technique which involves the replacement of
defective genes with healthy ones in order to treat genetic disorders. It
is an artificial method that introduces DNA into the cells of the human
body.
The first gene therapy was successfully accomplished in the year 1989.
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ABSTRACT

Gene therapy, a transformative branch of medical

science, offers unprecedented opportunities for
treating and potentially curing a myriad of genetic
disorders by manipulating or replacing faulty genes.
Grounded in the principles of precision medicine, gene
therapy involves the deliberate introduction, removal, or
modification of genetic material within a patient's cells,
 aiming to address the root causes of diseases at their
genetic core. The therapeutic approach encompasses a
diverse range of techniques, including the use of viral
vectors to deliver therapeutic genes, ex vivo and in vivo
strategies, and the revolutionary CRISPR-Cas9
technology for precise gene editing. Recent successes
in gene therapy, exemplified by the treatment of
conditions such as Severe Combined Immunodeficiency
(SCID) and inherited blindness, underscore its potential
to revolutionize the treatment landscape. Despite its
promise, gene therapy raises ethical concerns, ranging
from issues of informed consent and equitable access
to the risks associated with germline editing and
unintended consequences. Moreover, the high cost of
gene therapy introduces challenges related to
socioeconomic disparities in access. As the field
advances, ongoing research and interdisciplinary
collaboration are essential to address these ethical
considerations and unlock the full potential of gene
therapy, reshaping the future of medicine towards
personalized, targeted, and curative approaches.

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GENETIC DISORDER

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.

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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 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.

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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.

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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.

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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.

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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.

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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.

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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.

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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 biolistic
or gene gun.

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Benefits of Gene Therapy

Gene therapy holds the promise of treating and potentially

curing a wide range of genetic disorders by introducing,
removing, or
modifying genetic material within a person's cells. Some of
the key benefits of gene therapy include:
✓ Treatment of Genetic Disorders:
Gene therapy has the potential to treat and cure genetic
disorders by correcting or replacing faulty genes. This
includes conditions such as cystic fibrosis, sickle cell
anaemia, and muscular dystrophy.
✓ Precision Medicine:
Gene therapy allows for personalized and targeted
treatment, as it can be tailored to the specific genetic
makeup of an individual. This precision helps minimize side
effects and increases the effectiveness of the treatment.
✓ Reduced Need for Ongoing Treatment:
Unlike traditional treatments that may require lifelong
medication, gene therapy has the potential to provide a
one-time treatment that leads to a lasting or permanent
effect. This can significantly improve the quality of life for
patients.
✓ Treatment of Incurable Diseases:
Gene therapy offers hope for diseases that currently have
no cure. By addressing the root cause at the genetic level, it
opens up possibilities for treating conditions that were
previously considered untreatable.

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✓ Potential for Cancer Treatment:
Gene therapy is being explored as a treatment for certain
types of cancer. It may involve modifying a patient's own
immune cells to better target and destroy cancer cells,
 offering a promising approach to cancer treatment.
✓ Improved Treatment for Neurological Disorders:
Gene therapy is being investigated for treating
neurodegenerative disorders such as Parkinson's and
Alzheimer's disease. It holds the potential to slow or halt
disease progression by introducing genes that promote
neuronal survival or function.
✓ Treatment of Rare Diseases:
Gene therapy provides a targeted approach for treating rare
genetic disorders that affect a small percentage of the
population. This can make it economically viable for
pharmaceutical companies to develop treatments for these
fewer common conditions.
✓ Reduced Side Effects:
Compared to traditional treatments like chemotherapy,
gene therapy may have fewer side effects because it aims
to specifically target the affected cells without affecting
healthy cells.
Advancements in Biotechnology:
The development of gene therapy has driven advancements
in biotechnology and genetic engineering. This not only
benefits gene therapy itself but also contributes to
progress in related fields.

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Challenges of Gene Therapy
While gene therapy holds great promise, it also faces
several challenges that need to be addressed for its
successful and widespread application. Some of the
main challenges include:
• Safety Concerns: One of the primary challenges is ensuring
the safety of gene therapy. Introducing foreign genetic
material into a patient's cells can lead to unintended
consequences, such as triggering an immune response or
causing uncontrolled cell division, potentially leading to
cancer.

• Efficacy: Achieving consistent and high levels of gene

expression in the target cells is essential for the success
of gene therapy. Ensuring that the introduced genes are
effectively integrated into the patient's genome and
produce the desired therapeutic effect remains a
challenge.

• Delivery Methods: Efficient and targeted delivery of

therapeutic genes to the specific cells or tissues is a
significant hurdle. Developing effective delivery methods
that can safely and accurately transport genetic avoiding
off-target effects material to the target cells while is a
complex task.

• Immune Response: The body's immune system may

recognize the introduced genetic material as foreign and
mount an immune response against it. This can reduce the
effectiveness of gene therapy and may lead to adverse
reactions.

• Cost: Gene therapy treatments can be expensive, limiting

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accessibility for some patients. The high costs are often
associated with the complexity of the technology, research
and development expenses, and the relatively low patient
numbers for rare diseases.
• Off-Target Effects: Ensuring that gene editing or gene
addition occurs only in the intended cells is crucial.
Unintended modifications to other parts of the genome,
known as off-target effects, can have unpredictable and
potentially
harmful consequences.

• Long-Term Stability: Sustaining the therapeutic effect over

the long term is a challenge. Over time, the introduced
genes may lose their effectiveness, or the body's natural
mechanisms may climinate or silence them.

• Ethical Concerns: Gene therapy raises ethical questions

related to the potential for germ-line editing, where genetic
modifications can be passed on to future generations.
There are ongoing debates about the ethical implications of
making heritable changes to the human germline.

• Regulatory Approval: The regulatory pathway for gene

therapy is complex, and gaining approval from regulatory
agencies involves demonstrating both safety and efficacy.
Navigating these regulatory processes can be
time-consuming and costly.

• Limited Knowledge of Genetics: Our understanding of the

human genome is still incomplete, and many aspects of gene
function and regulation are not fully understood. This lack of
knowledge can pose challenges in designing precise and
effective gene therapies.

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Recent Advancements
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.
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.

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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.
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REFERENCES

❖http://en.wikipedia.org
❖Science daily
❖http://learn.genetics.utah.
edu ❖Youtube.com
❖http://www.trip2medi.com
❖https://www.sciencedirect.c
om/
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