“GENE THERAPY”

A Project submitted for the fulfillment of the requirements

For
CBSE CURRICULUM
SUBMITTED BY
Bhairavi Sharma
ROLL NO. STD. XII
Under the guidance
Of
Miss Mantasha A. Khan
To
NARAYANA E-TECHNO SCHOOL
 Certificate

This is to certify that the project entitled Gene Therapy is authentic work
carried out under my supervision as part of the CBSE curriculum of class
XII BIOLOGY and that it is as per the guidelines issued by the CBSE. To
the best of my knowledge, the project is original and a bona fide work
undertaken by Bhairavi Sharma.
 ACKNOWLEDGEMENT

I would like to express my sincere gratitude to Narayana E-techno School

for their invaluable support and guidance throughout the duration of this
project. Their expertise and encouragement have been instrumental in
shaping the outcomes of this work.
I am also deeply thankful to my supervisor, Mantasha A. Khan, for their
continuous encouragement, insightful feedback, and unwavering support.
Their mentorship has been pivotal in shaping my understanding and
approach to this research.
Furthermore, I extend my appreciation to my colleagues and friends for
their encouragement and assistance during the course of this project. Their
willingness to share resources and offer constructive criticism has enriched
the quality of this work.
 INDEX

TOPIC PAGE NO.

Gene therapy………………………………………………………………..1
Somatic gene therapy……………………………………………………….2
Germline gene therapy……………………………………………………...2
Gene Addition……………………………………………………………....2
Gene Editing………………………………………………………………..3
CRISPR Cas-9……………………………………………………………...4
Gene Silencing……………………………………………………………..5
Suicide Gene Therapy…………………………………………….………..6
Risk of gene therapy……………………………………………………….7
Conclusion…………………………………………………………………8
Self Reflection……………………………………………………………..9
Bibliography……………………………………………………………….10
 GENE THERAPY

Introduction
Genes contain DNA — the code that controls much of the body's form and function. DNA controls everything from
hair color and height to breathing, walking and digesting [Link] therapy aims to fix a faulty gene or replace it
with a healthy gene to try to cure disease or make the body better able to fight [Link] holds promise as a treatment
for a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and [Link] January
19, 1989, the director of the National Institutes of Health (NIH), Dr. James A. Wyngaarden, approved the first clinical
protocol to insert a foreign gene into the immune cells of persons with cancer. On September 14, 1990, W. French
Anderson and his colleagues at the NIH performed the first approved gene therapy procedure on a four-year-old girl
born with severe combined immunodeficiency (SCID). This initial trial was largely a success and over the next ten
years, 300 clinical gene therapy trials were performed on about 3000 individuals.
Types of Gene Therapy
Basically, there are two types of gene therapy:
Somatic Gene Therapy
This type usually occurs in the somatic cells of the human body. This is related to a single person and the only person
who has the damaged cells will be replaced with healthy cells. In this method, therapeutic genes are transferred into
the somatic cells or the stem cells of the human body. This technique is considered as the best and safest method of
gene therapy.

Germline Gene Therapy

It occurs in the germline cells of the human body. Generally, this method is adopted to treat the genetic,
disease-causing-variations of genes which are passed from the parents to their children. The process involves
introducing a healthy DNA into the cells responsible for producing reproductive cells, eggs or sperms. Germline
gene therapy is not legal in many places as the risks outweigh the rewards.

GENE ADDITION

Gene addition is used to treat conditions caused by a variation in a single gene. It is a one-time therapy that
involves the insertion of a functional copy of a missing or faulty gene into a person's cells by way of a viral or
non-viral genetically-engineered vector (delivery vehicle). Several steps involved in gene addition are:
Selection of Gene:The specific gene or DNA sequence that is to be added in the organism is identified. This gene
could be from the same species (for genetic modification or correction of defects) or from a different species (for
introducing new traits).
Isolation of Gene: To isolate the gene PCR (Polymerase Chain Reaction) primers are designed to flank the gene
of interest. Genomic DNA is then extracted from the organism or tissue using restriction endonuclease
enzymes(RENs), followed by PCR amplification. The resulting PCR products are analyzed using gel
electrophoresis to confirm the presence and size of the desired DNA fragment. Subsequently, the PCR product
containing the target gene is purified to remove contaminants, and its sequence is verified through sequencing.
Preparation of Vector: A vector is a carrier molecule that will transport the gene into the target organism's
[Link] are selected based on the DNA size. Some common vectors are Bacterial artificial chromosomes
(BACs),yeast artificial chromosomes (YACs) and viral vectors like retrovirus and adenovirus.
Gene Insertion into Vector: The isolated gene is inserted into the vector. This is typically done using molecular
cloning techniques such as ligation, which joins the gene and the vector DNA together with the use of DNA ligase.
Transformation or Transfection: Introduce the vector carrying the gene into the target cells of the organism.
This can be done through various methods such as bacterial transformation, viral transduction, electroporation, or
microinjection, depending on the organism and vector used.
Selection of Transformed Cells: Depending on the method used for gene delivery, selectable markers may be
included along with the gene of interest. These markers allow for the selection of cells that have successfully
incorporated the gene. Common selectable markers include antibiotic resistance genes or fluorescent proteins.
Integration into Host Genome: Once inside the target cells, the vector delivers the gene into the host genome.
The gene integrates into the genome through processes like homologous recombination or non-homologous end
joining, depending on the vector and target organism.
Expression of Gene: The integrated gene is transcribed and translated by the host cell's machinery, leading to the
production of the desired protein or trait.
Verification and Characterization: Confirm that the gene has been successfully integrated and is being
expressed in the host organism. This may involve various molecular biology techniques such as PCR, Southern
blotting, or protein analysis.
Propagation and Maintenance: Once cells or organisms with the desired gene addition have been identified and
verified, they can be propagated and maintained for further study or application.

GENE EDITING

Gene editing is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the
genome of a living organism. It is a site specific process performed using enzymes, particularly nucleases that
are made to target a specific DNA sequence, where they introduce cuts into the DNA strands, enabling the
removal of existing DNA and the insertion of replacement [Link] of the most well-known gene editing
techniques is CRISPR-Cas9, which uses a combination of a protein (Cas9) and RNA to target specific DNA
sequences and make changes to [Link] the advent of CRISPR-Cas9, two approaches were used to make
site-specific double-stranded breaks in DNA: one based on zinc finger nucleases (ZFNs) and the other based on
transcription activator-like effector nucleases (TALENs).
CRISPR-Cas9
CRISPR-Cas9 is currently the simplest, most versatile and precise method of gene manipulation. CRISPR-Cas9
was adapted from a naturally occurring genome editing system that bacteria use as an immune defense. When
infected with viruses, bacteria capture small pieces of the viruses' DNA and insert them into their own DNA in a
particular pattern to create segments known as CRISPR(Cultured Regularly Interspaced Short Palindromic
Repeats) arrays. The CRISPR arrays allow the bacteria to remember the viruses (or closely related ones). If the
viruses attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to
specific regions of the viruses' DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which
disables the [Link] gene editing there are CRISPR associated proteins which include Cas-9 which is a nuclease
enzyme.
Principle:
The CRISPR-Cas9 system consists of two key molecules that introduce a change in the DNA. These are:
● A nuclease enzyme Cas-9. This acts as a pair of molecular scissors that can cut the two strands of
DNA at a specific location so that bits of DNA can be added or removed.
● A piece of RNA called the guide RNA(gRNA). It consists of a small piece of pre designed RNA
sequence which is about 20 base pairs long. It is designed to find and bind to a specific sequence in
the DNA. It has bases complementary to that of the target DNA and thus guides the Cas-9 to the
right part of the genome.
The Cas-9 follows the gRNA to the same location and makes a cut across both the strands of the DNA. This break
can then be repaired by the cell leading to the desired genetic modification or disruption of the targeted gene.

Applications:
It finds various applications in treating genetic diseases such as sickle cell anemia,beta thalassemia and LCA( a
form of genetic blindness). Gene editing can also be used in agriculture to produce Genetically Modified
Organisms(GMOs).Researchers have explored using CRISPR-Cas9 to disrupt the HIV genome in infected cells,
potentially leading to a functional cure for HIV/AIDS. It has been investigated for its potential in cancer
immunotherapy, gene therapy, and understanding the genetic mechanisms underlying cancer development. It can
also induce phenotypic changes in an individual when used at the embryonic stage although this practice is legally
banned in many countries.
 GENE SILENCING

Silencing refers to blocking the gene’s expression that would otherwise cause an unwanted effect. Gene silencing
is a new technique that makes use of the body's natural processes to control diseases by suppressing the genes that
are associated with certain diseases. The main principle of gene silencing is based on RNA interference. RNA
interference is an inbuilt feature of the human body and which is designed to control the activity of genes and
defend the body against viruses. Gene silencing has controllable reversible effects which is highly advantageous
in case of any side effects. It is also highly targeted and specific, providing predictable results.

Principle:
RNA interference(RNAi) was a huge discovery in the field of molecular biology in the late 1990s. For this the
scientists Andrew Z. Fire and Craig Mello received the Nobel Prize for Medicine in 2006. The principle behind
gene silencing is to intervene in the process of gene expression before translation takes place. Small RNA
molecules such as interfering RNAs (iRNA) or microRNAs(miRNAs) are made to bind to complementary
sequences on target mRNA, leading to their degradation.
Epigenetic changes such as DNA methylation and histone modification can alter the structure of chromatin and
affect gene expression without changing the underlying DNA sequence. This can lead to gene silencing by
inhibiting the binding of transcription factors which in turn can lead to interference with the initiation or
elongation of RNA polymerase during transcription.

Applications:
Gene silencing is used not only to block unwanted gene expressions and induce viral resistance but also to
analyze unknown genes in sequenced [Link] is also being used to produce therapeutics to combat cancer,
infective diseases and neurodegenerative disorders. It is used in the agricultural industry to produce GMOs and to
lower the level of natural plant toxins.

SUICIDE GENE THERAPY

Suicide gene therapy was initially introduced as tumor chemosensitivity. It is a type of cancer treatment that
utilizes genetic engineering to selectively induce death of malignant tumor cells while the normal cells remain
unaffected. In this the gene is added into the somatic cells without replacing the abnormal gene. The therapy
involves introducing a gene encoding a prodrug-converting enzyme into cancer cells, which then converts an
inactive prodrug into a toxic drug specifically within the cancer [Link] is advantageous as this targeted approach
minimizes systemic toxicity and side effects associated with traditional chemotherapy.

Principle:
Introduction of Suicide Gene: A gene encoding a prodrug-converting enzyme, such as cytosine deaminase (CD),
is introduced into cancer cells. This is typically achieved using viral vectors, such as adenoviruses or retroviruses,
or non-viral methods like nanoparticles.
Administration of Prodrug: Once the suicide gene is expressed within the cancer cells, a non-toxic prodrug is
introduced artificially. The prodrug is designed to be selectively activated by the enzyme encoded by the suicide
gene.
Conversion to Toxic Drug: The prodrug-converting enzyme converts the inactive prodrug into its active form,
which is a cytotoxic drug. This conversion occurs specifically within the cancer cells that express the suicide
gene.
Selective Killing of Cancer Cells: The activated cytotoxic drug kills the cancer cells, leading to tumor regression
and destruction. Normal cells, which do not express the suicide gene, are unaffected by the prodrug and remain
unharmed.
Amplification of Therapeutic Effect: The cytotoxic effect of the activated drug may also induce programmed cell
death in neighboring cancer cells through bystander effects, further enhancing the therapeutic efficiency of suicide
gene therapy.

RISKS OF GENE THERAPY

Although gene therapy is an efficient method of treating cancer there are a numerous risks involved:
Off-Target Effects: One of the primary concerns with gene editing is the potential for off-target effects, where
the editing machinery introduces unintended changes to the genome at sites similar to the target sequence. These
off-target mutations could lead to unintended consequences, including the disruption of normal gene function or
the activation of oncogenes, potentially causing cancer or other adverse effects.
Immunogenicity and Immune Responses: Gene therapy approaches often involve the use of viral vectors to
deliver therapeutic genes into cells. However, the use of viral vectors can trigger immune responses in patients,
leading to inflammation, immune-mediated clearance of the vector, or neutralization of the therapeutic effect.
Pre-existing immunity to the viral vector or the development of immune responses against the transgene product
can limit the efficacy of gene therapy and pose safety risks.
Insertional Mutagenesis: In gene therapy, the integration of therapeutic genes into the host genome carries the
risk of insertional mutagenesis, where the insertion of foreign DNA into the genome may disrupt endogenous
genes or regulatory elements, potentially leading to oncogenesis or other adverse effects. This risk is particularly
relevant when integrating viral vectors such as retroviruses or lentiviruses.
Incomplete or Ineffective Treatment: Gene therapy and gene editing may not always achieve the desired
therapeutic outcome due to factors such as incomplete delivery of the therapeutic gene or editing machinery to
target cells, inadequate expression of the therapeutic gene, or insufficient correction of disease-causing mutations.
In some cases, this may result in partial or temporary improvements in disease symptoms or progression.
Ethical and Social ConsiderationS: The use of gene editing and gene therapy raises ethical and social concerns
related to the potential for misuse, unintended consequences, and equitable access to treatment. Questions
surrounding informed consent, privacy, equity, and the potential for enhancement also need to be addressed.
Long-Term Safety and Durability: Long-term safety and durability of gene editing and gene therapy
interventions remain areas of active investigation and concern. Assessing the long-term effects of genetic
modifications on patient health, monitoring for potential late-onset adverse effects, and ensuring sustained
therapeutic efficacy over time are important considerations for the clinical translation of these technologies.
 CONCLUSION

Biogenetics is a flourishing branch of science and gene modification and gene therapy are its important aspects.
It is an effective method to deal with diseases at a molecular level. For genetic disorders or in case of cancers
where medicinal drugs and surgeries might not be possible gene therapy provides a reliable alternative.
Although not fully developed, its various techniques help achieve efficient results through different methods.
It also helps find out the information about unknown genotypes helping scientists to find faster and more
reliable cure and treatment processes. It aims to personalize treatment based on an individual’s genetic makeup.
Diseases which were considered incurable now can be treated at least up to some extent. Though there are
doubts and ethical issues regarding it, gene therapy can be made more effective in the coming times with
advancing technology and strict rules to prevent its misuse.

SELF REFLECTION

I used to think that to treat a disease we must kill the microorganism causing it. I considered antibiotics and
surgeries to be the ultimate cure for any disorder. Now after learning about the cellular reactions occurring i
realized that by manipulating the genome of any microorganism we can not only prevent numerous diseases but
can also use those pathogens for our benefit. We don't need to prepare different medicines for different species
of microorganisms if we can manipulate its genetic material. Cell which is considered the unit of life might be
microscopic in its dimensions yet the reactions occurring inside it are vital for every aspect of [Link] project
made me appreciate that it is fascinating and pure luck that we are healthy and disease free every day. A small
change in the structure of DNA or a delay of small time in a reaction could lead to numerous problems in the
body. However I also realized that any disease which might seem incurable can be treated and overcomed by
various methods. If we can manipulate cell to our will it we can have immense control over the way our body
works.
 BIBLIOGRAPHY
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