Contents

· Abstract
· Introduction
· History of gene therapy
· Types of gene therapy
· Somatic gene therapy
· Germ line gene therapy
· Outcomes of gene therapy
· Functional classification
· Genetic disorders
· Targets for gene therapy
· Technologies used in gene therapy
· Gene Targeting
· Success cases of gene therapy
· Advantages & disadvantages
· Challenges
· Recent upcoming
· Conclusion
· References
ABSTRACT
Gene therapy is the introduction of genes into existing cells to prevent or cure a
wide range of diseases. It is a technique for correcting defective genes
responsible for disease development. The first approved gene therapy
experiment occurred on September 14, 1990, in US when Ashanti DeSilva was
treated for ADA-SCID. Gene therapy is designed to introduce genetic material
into cells to compensate for abnormal genes or to make a beneficial protein. If a
mutated gene causes a necessary protein to be faulty or missing, gene therapy
may be able to introduce a normal copy of the gene to restore the function of the
protein. A gene that is inserted directly into a cell usually does not function.
Instead, a carrier called a vector is genetically engineered to deliver the gene.
Certain viruses are often used as vectors because they can deliver the new gene
by infecting the cell. The viruses are modified so they can't cause disease when
used in people. Some types of viruses, such as retroviruses, integrate their
genetic material (including the new gene) into a chromosome in the human cell.
Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the
cell, but the DNA is not integrated into a chromosome.
Acknowledgment
I would like to express my special thanks of gratitude to my teacher (name) as
well as our principal (Name) who gave me the golden opportunity to do this
wonderful project on the topic “Gene therapy”, which also helped me in doing lot
of literature study and research and I came to know many new thing.

Secondly, I would also like to thank my parents and friends who helped me a lot
in finalizing this project within the limited time frame.

Divya Dak
INTRODUCTION
Gene therapy is an experimental technique that uses genes to treat or prevent
disease. In the future, this technique may allow doctors to treat a disorder by
inserting a gene into a patient’s cells instead of using drugs or surgery.
Researchers are testing several approaches to gene therapy, including:
⦁ Replacing a mutated gene that causes disease with a healthy copy of the
gene.
⦁ Inactivating, or “knocking out,” a mutated gene that is functioning
improperly.
⦁ Introducing a new gene into the body to help fight a disease.
Although gene therapy is a promising treatment option for a number of diseases
(including inherited disorders, some types of cancer, and certain viral infections),
the technique remains risky and is still under study to make sure that it will be
safe and effective. Gene therapy is currently being tested only for diseases that
have no other cures. An experimental technique for correcting defective genes
that are responsible for disease development.
The most common form of gene therapy involves inserting a normal gene to
replace an abnormal gene. It is intracellular delivery of genes to generate a
therapeutic effect by correcting an existing abnormality
TYPES OF GENE
THERAPY
⦁ Germ Line gene therapy
⦁ Somatic gene therapy

Germ line gene therapy

Therapeutic genes are transferred into the germ cells. It is heritable and passed
on to later generations. The result is permanent changes. Potential for offering a
permanent therapeutic effect for all who inherit the target gene. Possibility of
eliminating some diseases from a particular family.
E.g. Genes introduced into eggs and sperms.

Somatic Cell Gene Therapy

Therapeutic genes transferred into the somatic cells. Affects only the target cells
in the patient and is not passed to future generations. Short-lived because the
cells of most tissues ultimately die and are replaced by new cells. Transporting
the gene to the target cells or tissue is also problematic.
E.g. Introduction of genes into bone marrow cells, blood cells, skin cells etc.
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 unlocked 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.
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 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 must break the cell open
to release DNA along with other macromolecules such as RNA, proteins,
polysaccharides and 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 a 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
analyzed 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
are synthesized in vitro using two sets of primers (small chemically synthesized
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.
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 anemia, 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 possibilities
for treating conditions that were previously considered untreatable.
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 material to the target
cells while avoiding off-target effects 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.

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 localized, 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 leukemia (CLL), acute lymphocytic
leukemia (ALL), multiple myeloma, hemophilia 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
What is gene therapy https://ghr.nlm.nih.gov/primer/therapy/genetherapy
https://ghr.nlm.nih.gov/primer/therapy/genetherapy
Gene therapy information http://uniqure.com/patients/Gene-Therapy-
Information.pdf
http://en.wikipedia.org/wiki/Gene_therapy
Gene Therapy treatment http://www.trip2medi.com/treatmentCGeneTherapy.php
http://learn.genetics.utah.edu/content/tech/genetherapy/
Handbook on therapy http://ghr.nlm.nih.gov/handbook/therapy/
Cystic fibrosis http://cystic-fibrosis.emedtv.com/cystic-fibrosis/cystic-fibrosis-
gene-therapy.html
http://en.wikipedia.org
https://www.yourgenome.org/facts/what-is-gene-therapy
https://knowgenetics.org/gene-therapy/
https://www.sciencedaily.com/terms/gene_therapy.htm