BIOLOGY INVESTIGATORY

PROJECT
STUDY ON GENE THERAPY

     Class:-12th B       Subject :-Biology        Subject teacher:-  Mrs. Sunita purohit         BY:-Krish
 This is to certify that this “Biology Investigatory Project" on 
topic “Gene therapy” has been successfully completed by 
:- Krish of class – XIIth_B under the guidance of curriculum
of central board of secondary education (CBSE) leading to

CERTIFICATE the award of annual examination of the year 2022-2023.

Teacher in charge                                    External examiner
 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.
 Gene Therapy
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
So, if there is a flawed gene can we "fix" it? 1. Stay silent: ignore the genetic disorder and
malfunctions, cells that rely on that protein's
What are our options? nothing gets fixed.
function can't behave normally, causing
problems for whole tissues or organs. Medical
conditions related to gene mutations are
called genetic disorders.

If it is successful, gene therapy provides a way

to fix a problem at its source. Adding a
2. Try to treat the disorder with drugs or other corrected copy of the gene may help the
3. Put in a normal, functioning copy of the
approaches: depending on the disorder, affected cells, tissues and organs work
gene: if you can do this, it may solve the
treatment may or may not be a good long- properly. Gene therapy differs from traditional
problem!
term solution. drug-based approaches, which may treat the
problem, but which do not repair the
underlying genetic flaw.
TARGET FOR GENE
THERAPY
A disease to be targeted by gene therapy it must
satisfy the following conditions:
• The condition must result from mutations in one or more
genes
• 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.
• 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?
 • 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.
• 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. 
•  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.
• 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.
 Challenges
• Some the factors that have kept gene therapy from becoming an effective treatment for
genetic diseases are:
1) Short-lived nature of gene therapy -
o 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.
2) Immune response -
o 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.
3) 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.

4) 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.
CONCLUSION
• 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