RYAN INTERNATIONAL SCHOOL

I.S.C.,SURAT

NAME = NEHA PANDEY

CLASS = XII
ROLL NO. = 22
SUBJECT = BIOLOGY
TOPIC = GENE THERAPY
TEACHER INCHARGE = MR. PRATHAMESH PATEL
ACADEMIC YEAR = 2022-2023
 Bonafide Certificate

This is to certify that the project report entitled “GENE

THERAPY” submitted by “NEHA PANDEY” considered as a
part of the exam of ISC conducted by CISCE is a bonafide
record of the work carried out under our guidance and
supervision at RYAN INTERNATIONAL SCHOOL, ISC, SURAT.

This project is evaluated by us on:

INTERNAL EXAMINER EXTERNAL EXAMIMER

HEAD OF INSTITUTION SCHOOL STAMP

 ACKNOWLEDGEMENT
I would like to express my special thanks to our school Ryan
International School and principal miss Ms Sandhya Shaji and my
subject teacher Mr. PRATHAMESH PATEL who have given me the
golden opportunity to do this wonderful project which also helped
me in doing a lot of research and I came to know about so many new
things. Secondly I would like to also thank my parents and friends
who helped and supported me in finishing the project.
 INDEX
Sr. No. Topic Page no.
1. INTRODUCTION
2. GEND THERAPY
3. TARGETS FOR GENE THERAPY
4. CHOOSING THE BEST VECTOR
Introduction
Diseases
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 civilisation interchangeably)
are diseases that appear to increase in frequency as
countries become more industrialised 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 eternal agents. Also
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 the
gene therap.
 

Gene Therapy
We can think of a medical condition or illness as a “ broken
window” . Many medical conditions result from flaws , all mutation,
in one or more persons gene’s. Mutation causes the protein in
encoded by that gene to malfunction when a protein malfunctions,
cell that rely on that protein function can’t behave normally,
causing problems for whole tissue or organ. medical condition
related to gene mutation are called genetic disorder.
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 Disorders 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.
Targets for Gene Therapy
But now a question arises , which disorders or diseases can be
target using gene therapy ? many disorders or medical condition
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 mutation is one or more gene.
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 mutation 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 tissue are affected?
. what role does the protein encoded by the gene play within
the cells of that tissue?
. Exactly how do mutation in the gene affect the proteins
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 is the mutated gene encodes a protein that prevents the
normal protein formed doing its job? Mutated gene 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?
 That 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 organisms. Finding the
right gene to insert usually draws on years of scientific
research into the Identity and function of useful geans . Once
that is known the DNA needs to be cut at specific location 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 in
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 macro
molecule 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
protease. Other molecules can be removed by appropriat
treatments and purified DNA ultimately precipitates out the
 addition of chilled ethanol. This can be seen as collection of
fine threads in the suspension.

Cutting of DNA
Restriction enzyme digestion are performed by incubating purified
DNA molecules with the restriction enzymes, at the optimal
conditions for that specific enzyme . The cutting of DNA by restriction
endonucleases result in the fragments of DNA . These fragments
can be separated by a technique known as gel electrophoresis. Since
DNA fragments and negatively charge 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 and 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 synthesized in vitro using two sets of
primers (small chemically synthesized oligonucleotides that are
complementary to the region of DNA ) and the enzyme DNA
polymerase . The enzyme explain the primary using the nucleotide
provide in the reaction and the genomic DNA as templates. 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 to gene
therapy .
Gene delivery includes :
1 Targeting the right cells .

2 Activating the gene . A gene journey is not over when it enter

the cells . It must go to the cells 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 produce 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 cells
genetic material, or that gene finds another way to survive in the
nucleus without being rejected .

4 AVODDING harmful side effects. anytime 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
deliver vehicles ,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 first
miles to address the unique features of the disorder . we have learnt
the lesson, of transferring into plants and animals from bacteria and
viruses, which have known this for ages- how to deliver genes to
transform eukaryotic cell and force them to do with what the
bacteria or viruses want .
Part of the challenging gene therapy using 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, scientist looks to viruses .why reinvent
and the wheel is there 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 by engineered to target specific types of
cells .
- They can be modified so that they can’t replicate and destroy the
cell .
General drawback of viral vectors:
A virus can’t “expand” to fit a piece of genetic material larger than it
is naturally built to carry . Theirfor some maybe to 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 causes an immune response.
Non viral vectors
Although viruses can effectively delivered 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 a immune responses.
Non- viral vectors a typically circular DNA molecules, also known as
plasmid. In nature, bacteria use plasmids to transfer genes from cell
to cell.
Vector used at present, are engineered in such a way that they helps
easy linking of foreign DNA and selection of recombination 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
injection into the nucleus of an animal cell . In other method ,
suitable for plants, cell or bombarded with high velocity micro
particles of gold or tungsten coated with DNA in the method known
as biolistic or gene gun.

Delivery to specific tissues

Delivering genes to specific tissues within a patient’s body cab be
very difficult. Delivering genes into a group of cell in a patient’s body
ca be done in one of two ways .
The first way is to inject the vector into the body and specifically
target affected cells . This is called in Vivo approach the second way,
called ex Vivo is to deliver the gene to cell while they are outside the
body :by :
. Isolating the desired cell from the body .
. culturing the cell in a Petri dish in the laboratory .
. Delivering the genes to cells ,(using one of the vector options
described on this page ) activating them , and making sure that the
cells integrated them properly .

Case study- cystic fibrosis

The Disease – A Genetic Disorders
Cystic fibrosis (CF) , Alan 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 and chloride and sodium across and epithelium,
leading to thick ,viscous secretions, why preventing the cilia from
clearing debris which cause symptoms such as coughing , poor
digestion and increased vulnerability to infection.

CF is caused by mutation in the gene for the proteins cystic fibrous

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 regulation of
components of sweet digestive fluids, and mucus (CFTR) regulates
the movements of chloride and sodium ions across epithelium
membrane such as the alveolar epithelial located in the lungs. Since
all of the cell of CF patient have the defective protein, large
quantities thick , sticky mucus build up throughout the lungs and
other organs. This result in the severity of symptoms seen in CF
patients.
Is It A Good Target For Gene Therapy?
To check this some question 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 CFTR gene should fix the problem.
. Is the 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 surface 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 is a perfect diseases to be treated by
gene therapy .

Choosing vectors
The vector that are most suitable for gene therapy are :

Retrovirus
Retrovirus are enveloped viruses that replicate in a host cell through
the process of reserve transcription. it is a single standard RNA virus
that stores its nucleic acid in the form of an mRNA genome targets .
Once inside the host cell cytoplasm the virus uses it own reverse
transcriptase enzyme to produce DNA from its RNA genome, the
reserve of the usual pattern, thus retro (backwards) . This new DNA
is then incorporated into the host cells genome by an integrase
enzyme, at which point retroviral DNA is referred to as a provirus .
The host cells then treats the viral DNA as part of it’ own genome ,
translating and transcribing the viral genes along with the cells own
genes , producing the proteins required to assemble new copies of
the virus .
One drawbacks of retroviruses , such as the Moloney retrovirus
involves the requirement for cells to be actively dividing the
transduction . As a result , cells such as neurons on very resistant to
infection and transduction by retroviruses.
 But the airway cells are affected by diseases cystic fibrosis and must
be targeted divide infrequently. Hence Retrovirus is not a suitable
vector for this disease.

Adenovirus
Adenoviruses (members of the family Adenoviridae) are
mediumsized (90–100 nm),non enveloped (without an outer lipid
bilayer) viruses with anicosahedralnucleocapsid containing a double
stranded DNA genome.
They have a broad range of vertebrate hosts and have been found to
cause a wide range of illnesses, from mild respiratory infections in
young children to life-threatening multi-organ disease in people with
aweakened immune system.
But they can cause/induce an immune response in the humanbody
hence not suitable for gene delivery.

Herpes Simplex Virus

Herpes simplex viruses, also known as Human herpes virus, are
members of the herpes virus family, Her pesviridae, that infect
humans. They can be spread when an infected person is producing
and shedding the virus. Herpes Simplex can be spread through
contact with saliva, such as sharing drinks.
But these viruses only affect the cells of the nervous system and
cannot infect the air way cells and hence not suitable.
Adeno-Associated Viruses
Adeno-associated virus(AAV) is a small virus which infects humans
and some other primate species. AAV is not currently known to
cause disease and consequently the virus causes a very mild immune
response. AAV can infect both dividing and quiescent cells and
persist in an extra chromosomal state without integrating into the
genome of the host cell. Despite its few disadvantages these features
make AAV a very attractive candidate for creating viral vectors for
gene therapy, and for the creation of isogenic human disease models
Hence it is the best choice for gene delivery in the case of Cystic
Fibrosis.

History of Cystic Fibrosis Gene Therapy

Gene therapy for cystic fibrosis began in 1990, when scientists
successfully corrected faulty cystic fibrosis trans membrane
conductance regulator (CFTR) genes. They did this by adding normal
copies of the gene to laboratory cell cultures.

1993
In 1993, the first experimental CF gene therapy treatment was given
to a patient with cystic fibrosis. Researchers modified a common cold
Adenovirus to act as a delivery vehicle by carrying normal genes to
the CFTR cells in the nasal passages. Researchers chose nasal
passages as the site of delivery because they are easier to access and
measure gene activity than the lung airway. Later trials delivered the
vector to patients’ lung airways.
In the earlier trials, it had looked like the virus had entered cells and
that the CTFR gene was working. But later trials with different
patients showed levels of VFTR gene activity that were too low to
make any difference. Researchers came to think that the adenovirus
can’t easily enter airway cells, especially in the low doses that were
being given. In the earlier trials, they speculated, gene activity
resulted from the damage to the cells during delivery allowing the
virus to enter easily .Hence when higher doses of the virus were
tried, the immune system of the patient s started mounting immune
responses and fighting off the virus. This caused a blockage in the
trials until 1998.

1998
Trials using Adeno-associated virus to deliver the CTFR gene began in
1998. Unlike the adenovirus, the Adeno-associated virus caused no
immune response or adverse side effects in patients.
But unlike the researchers’ predictions, the adeno-associated virus
did not enter cells efficiently and integrate into calls’ genomic DNA.
They produced only low and fleeting amounts of CFTR gene activity.
Researchers are still working to figure out what caused the viruses to
fail.
But because it is safe, the virus – as we predicted earlier – holds
promise for being a good way to deliver the CFTR gene to patients’
airway cells. But researchers need to learn more about how the virus
infects cells in order or make it an effective delivery method.

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.
•Multi gene 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. Multi gene or multifactorial disorders such as these
would be especially difficult to treat effectively using gene therapy.
Issues regarding Gene Therapy
What are the possible implications of gene therapy research to
society? All of us -researchers, policy makers and the public - have a
responsibility to explore the potential effects of gene therapy
research on our lives so that we can make informed decisions.
For each new application of gene therapy research, we must
consider:

• What are the benefits?

• What are the risks?

• Whom will the technology help? Who will it potentially hurt?

• What does gene therapy mean for us?

There are several types of issues to consider as we think about gene

therapy:

• Ethical issues ask us to consider the potential moral outcomes

of gene therapy research.

• Legal issues require researchers and the public to help policy

makers decide whether and how gene therapy research should
be regulated by the government.

• Social issues involve the impact of gene therapy research on

society as a whole.
Some questions to ponder

• When should gene therapy be used? Should it be used to treat

critically ill patients? Should it be used to treat babies and
children?

• What effect would gene therapy have on future generations if

germline (reproductive) cells were genetically altered? How
might this alteration affect human variation?

• Who should decide what are "good" or "bad" uses of genetic

modifications? How do you define "normal" with regard to
human beings?

• What if we could alter human traits not associated with

disease? Would it be okay to use gene therapy to improve or
enhance a person's genetic profile?

• Who will have access to gene therapy, treatments and long

term follow-ups? Will gene therapy and genetic enhancements
create an advantage for those who can afford it?

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.

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 investement in the field.
BIBLIOGRAPHY

.www.wikipedia.com
.www.byjus.com
. www.google .com
.www. Scribd . Com